Multi-functionalized polysaccharide compounds and use thereof for targeting the cation-independent mannose 6-phosphate receptor

ABSTRACT

The subject matter of the present invention is a compound characterized in that it has the general formula (I): in which P 1 , X, n, A, L and L 1  are as defined in Claim  1 . The present invention also relates to a process for preparing said compounds (I) and to the use thereof for targeting the cation-independent mannose 6-phosphate receptor (CI-M6PR). The subject matter of the invention is also a conjugate of formula (III): in which P 1 , X, n, A, L and L′ 1  are as defined in Claim  6 , and the use thereof: —in a method for therapeutic treatment of the human or animal body, in particular chosen from enzyme replacement therapy, photodynamic therapy or cancer treatment, and/or—in a method of diagnosis, in particular of diseases or of ailments associated with an increase or with a decrease in CI-M6PR expression.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

The present patent application is filed pursuant to 35 U.S.C. § 371 as aU.S. National Phase Application of International Patent Application No.PCT/FR2016/052339 filed Sep. 15, 2016, claiming the benefit of priorityto French Patent Application No. 15 58806 filed Sep. 18, 2015. TheInternational Application was published as WO 2017/046535 on Mar. 23,2017. The contents of each of the aforementioned patent applications areherein incorporated by reference in their entirety.

The present invention relates to novel multi-functionalizedpolysaccharide compounds, and more particularly multi-functionalizeddi-, tri- or tetramannocide compounds. The invention also relates to theprocess for preparing said compounds, and to the use thereof fortargeting the cation-independent mannose 6-phosphate receptor (CI-M6PR).

The cation-independent mannose 6-phosphate receptor (CI-M6PR) is aubiquitous receptor which is present both in the cytoplasm and in themembrane of cells. Its main role is to provide the transportation ofnewly synthesized lysosomal enzymes from the trans-golgi network to thelysosomes where they are active. It also participates in the endocytosisof lysosomal enzymes secreted by the cells. This endocytosis by CI-M6PRallows the internalization of compounds bearing mannose 6-phosphate(M6P) residues into the cell and the trafficking thereof to thelysosomes.

CI-M6PR thus plays a crucial role in the treatment of lysosomal overloaddiseases (rare disease), and more particularly for enzyme replacementtherapy. Enzyme replacement therapy is a therapy used successfully totreat these rare diseases which are characterized by a deficiency of aspecific lysosomal enzyme. It consists of the administration of therecombinant lysosomal enzymes which will be directed to the lysosomesvia CI-M6PR by virtue of the M6P residues present at the end of theirglycosylated chains.

Furthermore, CI-M6PR is overexpressed in certain cancers¹, which makesit an advantageous target for the development of targeted treatments,thus limiting toxicity of healthy cells.

Such therapies can be obtained by the functionalization, for example, ofnanoparticles encapsulating an active ingredient and surface-graftedwith mannose 6-phosphate (M6P) residues.

However, the major drawback of mannose 6-phosphate (M6P) is thesensitivity of its phosphate function to hydrolysis by the phosphatasespresent in all the organs and in serum. M6P is then dephosphorylated andis no longer recognized by CI-M6PR. The stability of this recognitionmarker is therefore a determining element for its use in thetransportation of bioactive molecules.

The isosteric analogs of M6P do not have the drawback of degradation byphosphatases^(2,3,4). The ligation of these isosteric analogs of M6P tothe oligosaccharide part of lysosomal enzymes makes it possible toimprove the efficiency of these enzymes for applications in enzymereplacement therapy⁵. The use of these isosteric analogs also allowsvery effective targeting of cancer cells of prostate tumorsoverexpressing CI-M6PR and also their treatment¹.

In order to improve the affinity for CI-M6PR, these same analogs havebeen synthesized in dimannose series with a glycosidic bond of α(1,2)type. The disaccharide 6-P-Man-α(1,2)-Man has an affinity for CI-M6PRwhich is greater than that of the monosaccharide M6P⁶. The alpha-1,2linkage between the two mannose residues is very important for obtaininggood affinity with respect to CI-M6PR. The alpha-1,3 or alpha-1,4 oralpha-1,6 linkages result in a lower affinity.

The 6-P-Man-α(1,2)-Man disaccharide has, however, the drawback of notbeing stable in the blood. This is because the phosphate function of the6-P-Man-α(1,2)-Man disaccharide undergoes hydrolysis by the phosphatasespresent in serum. After dephosphorylation by the hydrolases of theserum, the disaccharide formed, Man-α(1,2)-Man, is not recognized byCI-M6PR which is the biological target.

There remains at this time the need to find new isosteric analogs of M6Pwhich have an improved affinity for CI-M6PR, and which also are stablein physiological fluids.

One of the objectives of the present invention is therefore to providenovel isosteric analogs of M6P which have a very high affinity forCI-M6PR.

The expression “isosteric analog of M6P” is intended to mean a syntheticchemical compound having the same biological activity as M6P, butimproved stability.

Another objective of the invention is to provide novel isosteric analogsof M6P which are stable in physiological fluids.

Another objective of the invention is to provide novel isosteric analogsof M6P which have improved affinity for CI-M6PR compared with theisosteric analogs of M6P that are described in applicationPCT/EP2010/05950⁵.

The inventors have thus imagined novel multi-functionalized di-, tri- ortetramannocide compounds, that is to say compounds that bear variousfunctional groups, said functional groups being capable of forming,respectively, one (or more) bond(s) with CI-M6PR and one (or more)bond(s) with a compound of interest Y.

The difficulty encountered with the compounds provided in the inventionis to find an advantageous way to synthesize them, namely in particulara way which is simple to carry out, efficient (good yield) andinexpensive.

Another objective of the invention is therefore to find a process forpreparing multi-functionalized di-, tri- or tetramannocide compoundswhich is advantageous, namely in particular which is simple to carryout, efficient and inexpensive.

Indeed, the synthesis of di-, tri- or tetrasaccharide compounds is morecomplex to carry out than that of monosaccharide compounds.

A subject of the present invention is a compound characterized in thatit has the general formula (I):

in which:

-   -   n is an integer ranging from 1 to 3,    -   represents a single bond or a double bond,    -   each of the P¹ represents, independently of one another, H, or        an acid-labile, base-labile, hydrogen-labile, photo-labile,        halogen-labile protecting group, in particular chosen from        trimethylsilyl (TMS: (CH₃)₃Si—), tert-butyldimethylsilyl (TBDMS:        tBuMe₂Si—), benzyl (Bn: C₆H₅CH₂), para-methoxybenzyl (PMB:        4-CH₃OC₆H₅CH₂—), ortho-dinitrobenzyl (o-NO₂C₆H₅CH₂—), acetyl        (Ac: CH₃CO—), benzoyl (Bz: C₆H₅CO—) or CF₃CO— (trifluoroacetyl),    -   X represents:        -   when            is a single bond:            -   the phosphonate group:

-   -   -   -   the fluorophosphonate group:

-   -   -   -   the difluorophosphonate group:

-   -   -   -   the carboxylate group:

-   -   -   -   the malonate group:

-   -   -   when            is a double bond:            -   the phosphonate group:

-   -   -   -   the carboxylate group:

with Z representing, independently of one another, H; a C₁-C₅ alkylchosen in particular from methyl (Me: CH₃—), ethyl (Et: C₂H₅) orisopropyl (iPr: (CH₃)₃C—); 2,2,2-trifluoroethyl (CF₃CH₂—); C₆H₅CH₂—;phenyl (Ph: C₆H₅—); (CH₃)₃Si—; an alkali metal chosen from Na, Li or K;an ammonium NH₄;

-   -   A represents a divalent radical chosen from —O—, —S—, —NH—,        —CH₂—;    -   L represents:        -   —H; —NH₂; —(CH₂)_(n1)—CH═CH₂ or —(CH₂)_(n1)—C≡CH with n₁            representing an integer ranging from 0 to 4, then in each of            these cases, L₁ is absent,        -   a substituted or unsubstituted, linear or branched,            saturated divalent hydrocarbon-based radical having from 1            to 30 carbon atoms, a substituted or unsubstituted, linear            or branched, unsaturated divalent hydrocarbon-based radical            having from 2 to 30 carbon atoms,        -   a saturated or unsaturated divalent hydrocarbon-based            radical as defined above, in which one or more —CH₂—,            —CH═CH— and/or —C≡C— groups of the saturated or unsaturated            hydrocarbon-based radical is (are) replaced, independently            of one another, with an ether (—O—) group; amine (—NH—)            group; alkylamine (—NR₁—) group with R₁ representing a C₁-C₅            alkyl; thioether (—S—) group; amide (—CO—NH—) group;            carbamate (—NH—CO—O—) group; oxime —O—N═CH— group;            acylhydrazone —CO—NH—N═CH— group; a substituted or            unsubstituted, saturated or unsaturated, cyclic or            heterocyclic system;    -   L₁ represents:

—(CH₂)_(n1)—CH═CH₂; —(CH₂)_(n1)—C≡CH; —(CH₂)_(n1)—N₃; —(CH₂)_(n1)—SH;—(CH₂)_(n1)—NH₂; —(CH₂)_(n1)—N═C═O; —(CH₂)_(n1)—N═C═S; —(CH₂)_(n1)—NHR₁;—(CH₂)_(n1)—NR₁R₂; —(CH₂)_(n1)-A₁-NH₂; —(CH₂)_(n1)-A₁-NHR₁;—(CH₂)_(n1)-A₁-NR₁R₂; —(CH₂)_(n1)—NHCO—CH₂Hal; —(CH₂)_(n1)—COZ₁;—(CH₂)_(n1)-A₁COZ₁; —(CH₂)_(n1)—O—N═CH₂; —(CH₂)_(n1)—CO—NH—N═CH₂;—(CH₂)_(n1)—H; a substituted or unsubstituted, saturated or unsaturated,cyclic or heterocyclic system; a halogen chosen from F, Cl, Br or I;with

-   -   n₁ as defined above,    -   R₁ and R₂ representing, independently of one another, a C₁-C₅        alkyl,    -   A₁ representing —O—, —NH—,

Hal representing Cl, Br or I;

Z₁ representing —OH, —OR₁, —NH—NH₂, —NH—NR₁R₂, with R₁ and R₂ as definedabove, a halogen chosen from F, Cl, Br or I.

By way of examples of a linear or branched, saturated divalenthydrocarbon-based radical having from 1 to 30 carbon atoms, mention mayin particular be made of:

—CH₂—CH₂—; —CH₂—(CH₂)_(m)—; —(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—;

—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—;—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m);

—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m);—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m)—;

with m representing, independently of one another, an integer rangingfrom 0 to 30 with the condition that the length of the mainhydrocarbon-based chain does not exceed 30 carbon atoms, and C₁-C₇representing an alkyl having from 1 to 7 carbon atoms. By way of exampleof C₁-C₇ alkyl, mention may in particular be made of methyl, ethyl,propyl, isopropyl, butyl, isobutyl, pentyl, hexyl or heptyl.

A linear or branched, unsaturated divalent hydrocarbon-based radicalhaving from 2 to 30 carbon atoms denotes a hydrocarbon-based radicalcomprising one or more carbon-carbon double bonds and/or one or morecarbon-carbon triple bonds. By way of examples of an unsaturateddivalent hydrocarbon-based radical, mention may in particular be madeof:

—CH═CH—; —(CH₂)_(m)—CH═CH—(CH₂)_(m)—;

—(CH₂)_(m)—CH═CH—(CH₂)_(m)—CH═CH—(CH₂)_(m)—;

—(CH₂)_(m)—CH═CH—CH(C₁-C₇)—(CH₂)_(m)—;

—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—CH═CH—CH(C₁-C₇)—(CH₂)_(m)—;

—(CH₂)_(m)—CH(C₁-C₇)—CH═CH—(CH₂)_(m)—;

—(CH₂)_(m)—CH(C₁-C₇)—CH═CH—(CH₂)_(m)—CH═CH—(CH₂)_(m)—;

—(CH₂)_(m)—CH═CH—C(C₁-C₇)₂—(CH₂)_(m)—;

—(CH₂)_(m)—CH═CH—C(C₁-C₇)₂—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m)—CH═CH;

—(CH₂)_(m)—CH═CH—C(C₁-C₇)₂—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—CH═CH;

—C≡C—; —(CH₂)_(m)—C≡C—(CH₂)_(m)—;—(CH₂)_(m)—C≡C—(CH₂)_(m)—C═C—(CH₂)_(m)—;

—(CH₂)_(m)—CH═CH—CH(C₁-C₇)—(CH₂)_(m)—C≡C—(CH₂)_(m)—CH(C₁-C₇)—;

—(CH₂)_(m)—CH═CH—C(C₁-C₇)₂—(CH₂)_(m)—C≡C—(CH₂)_(m)—C(C₁-C₇)₂—(CH₂)_(m)—;

—(CH₂)_(m)—C≡C—CH(C₁-C₇)—(CH₂)_(m)—C≡C—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m);

—(CH₂)_(m)—C≡C—C(C₁-C₇)₂—(CH₂)_(m)—CH═CH—(CH₂)_(m)—CH(C₁-C₇)—;

—(CH₂)_(m)—CH═CH—C(C₁-C₇)₂—(CH₂)_(m)—CH(C₁-C₇)—(CH₂)_(m)—C≡C—CH═CH—(CH₂)_(m)—;

with m as defined previously.

According to the invention, the term cyclic or heterocyclic “system”denotes, as appropriate, the monovalent or divalent radical originatingfrom a saturated or unsaturated, cyclic or heterocyclic compound, assuch.

Thus, when L₁ represents a cyclic or heterocyclic system, it will morespecifically be a monovalent radical originating from a saturated orunsaturated, cyclic or heterocyclic compound.

When L represents a saturated or unsaturated hydrocarbon-based radicalin which one or more —CH₂—, —CH═CH— and/or —C≡C— groups is (are)replaced, independently of one another, with a saturated or unsaturated,cyclic or heterocyclic system, then said cyclic or heterocyclic systemis a divalent radical originating from a saturated or unsaturated,cyclic or heterocyclic compound.

By way of example of a saturated or unsaturated, cyclic or heterocycliccompound, mention may in particular be made of azetidine, oxetane,thietane, pyrrole, pyranose, furanose, furan, pyrroline,tetra-hydrofuran, thiophene, tetra-hydrothiophene, pyrazole, imidazole,oxazole, isoxazole, pyrazoline, imidazoline, pyrazolidine,imidazolidine, dioxolane, thiazole, isothiazole, thiazolidine,isoxazolidine, triazole, oxadiazole, thiadiazole, thiosuccinimide,tetrazole, pyridine, naphthyridine, phthalimide, pyran, dihydropyran,piperidine, pyridazine, pyridinium, pyrimidine, purine, pyrazine,pteridine, oxazine, dioxine, piperazine, maleimide, morpholine, dioxane,thiazine, thiomorpholine, oxathiane, dithiane, triazine, trioxane,thiadiazine, dithiazine, trithiane, 3-cyclobutene-1,2-dione,cyclobutane, cyclobutene, cyclopentane, cyclopentene, cyclohexane,cyclohexene, cycloheptane, cycloheptene, benzene, toluene, naphthalene,indene, indane, indolizine, indole, benzofuran, indoline,benzothiophene, indazole, benzimidazole, benzthiazole, tetraline,quinoline, chromene, chromane, cinnoline, quinazoline, quinoxaline andphthalazine.

According to the invention, and as previously indicated, said linear orbranched, saturated or unsaturated divalent hydrocarbon-based radical,and said saturated or unsaturated, cyclic or heterocyclic system, mayalso be substituted with one or more substituents chosen from C₁-C₇alkyl, C₂-C₇ alkenyl, and C₂-C₇ alkynyl, aryl or with functional groups.

By way of example of functional groups, mention may in particular bemade of alcohol, amine, amide, ketone, ester, ether, thioether orcarboxylic acid functions.

The term “C₂-C₇ alkenyl” denotes a linear or branched hydrocarbon-basedradical having 2 to 7 carbon atoms and comprising one or morecarbon-carbon double bonds.

The term “C₂-C₇ alkynyl” denotes a linear or branched hydrocarbon-basedradical having 2 to 7 carbon atoms and comprising one or morecarbon-carbon triple bonds.

The term “aryl” denotes a mono-, bi- or tricyclic aromatichydrocarbon-based system having from 6 to 18 carbon atoms. By way ofexample, mention may be made of phenyl (C₆H₅), benzyl (C₆H₅CH₂),phenethyl (C₆H₅CH₂CH₂), tolyl (C₆H₄CH₃), xylyl (C₆H₃(CH₃)₂), benzylidene(C₆H₅CH═CH), benzoyl (C₆H₅CO), biphenyl (or diphenyl) (C₁₂H₉) ornaphthyl (C₁₀H₇).

According to one embodiment of the invention, in the compound of formula(I) as defined above:

L represents:

-   -   —NH₂, —(CH₂)_(n1)—CH═CH₂ or —(CH₂)_(n1)—C≡CH, with n₁ as defined        above (integer ranging from 0 to 4), then in each of these        cases, L₁ is absent,    -   a substituted or unsubstituted, linear or branched, saturated        divalent hydrocarbon-based radical having from 1 to 10 carbon        atoms, a substituted or unsubstituted, linear or branched,        unsaturated divalent hydrocarbon-based radical having from 2 to        10 carbon atoms,    -   a saturated or unsaturated, divalent hydrocarbon-based radical        as defined above, in which one or more —CH₂—, —CH═CH— and/or        —C≡C— groups of the saturated or unsaturated hydrocarbon-based        radical is (are) replaced, independently of one another, with:        -   an —O— group; —NH— group; —S— group; —CO—NH— group;            —NH—CO—O— group; and/or        -   a cyclic or heterocyclic system chosen from:

L₁ represents:

—(CH₂)_(n1)—CH═CH₂; —(CH₂)_(n1)—C≡CH; —(CH₂)_(n1)—N₃; —(CH₂)_(n1)—SH;—(CH₂)_(n1)—NH₂; —(CH₂)_(n1)—N═C═O; —(CH₂)_(n1)—N═C═S;—(CH₂)_(n1)—O—NH₂; —(CH₂)_(n1)—NHCO—CH₂Hal; —(CH₂)_(n1)—COOH;—(CH₂)_(n1)—COOR₁; —(CH₂)_(n1)—CO—NH—NH₂; —(CH₂)_(n1)—H; a halogenchosen from F, Cl, Br or I;

-   -   a cyclic or heterocyclic system chosen from:

with n₁, R₁ and Hal as defined above.

By way of examples of compounds of formula (I) as defined above, mentionmay be made of those for which:

-   -   the substituent L is chosen from:        -   —CH═CH₂, —C≡CH or —NH₂, then in each of these cases, L₁ is            absent;        -   —CH₂—; —CH₂—CH₂—; —(CH₂)₃—; —(CH₂)₄—; —(CH₂)₅—;            —CH₂—CH₂—O—CH₂—CH₂—; —(CH₂—CH₂—O)₂—CH₂—CH₂—;            —CH₂—CH₂—S—CH₂—CH₂—; —CH₂—CH₂—S—CH₂—CH₂—O—CH₂—CH₂—;

and

-   -   the substituent L₁ is chosen from:        -   —CH═CH₂; —C≡CH; —N₃; —SH; —NH₂; —N═C═O; —N═C═S; —O—NH₂;            —NHCO—CH₂Cl; —COOH; —COOR₁; —CO—NH—NH₂, a halogen chosen            from F, Cl, Br or I,        -   a cyclic or heterocyclic system chosen from:

-   -   with R₁ as defined above.

A compound of formula (I) in which n is equal to 1 is a disaccharide,and more particularly a multi-functionalized di-mannoside correspondingto the formula:

A compound of formula (I) in which n is equal to 2 is a trisaccharide,and more particularly a multi-functionalized tri-mannoside correspondingto the formula:

A compound of formula (I) in which n is equal to 3 is a tetrasaccharidecompound, and more particularly a multi-functionalized tetramannosidecorresponding to the formula:

The invention also relates to a process for preparing a compound offormula (I) as defined above, characterized in that the startingcompound used is a compound corresponding to the formula (II):

in which P¹, n, A, L and L₁ are as defined above.

These compounds are very advantageous in that they make it possible inparticular to prepare compounds of formula (I) in a limited number ofsteps, and with a good yield.

A subject of the invention is therefore also a compound corresponding tothe general formula (II):

in which P¹, n, A, L and L₁ are as defined above.

The preparation of the compound of formula (II) is carried out byreaction between a glycoside acceptor (IV) or (IVa) with a glycosidedonor (V), according to one of the reaction schemes below:

The compounds of the invention of formula (I) are particularlyadvantageous because, on the one hand, they make it possible to targetCI-M6PR with a very high affinity and, on the other hand, they arecapable of forming covalent bonds at their group LL₁ with a large numberof compounds of interest {circle around (Y)}.

The present invention also relates to a conjugate formed between acompound of interest {circle around (Y)} and the compound of formula(I).

A subject of the invention is thus also a conjugate characterized inthat it corresponds to general formula (III):

in which X, P¹, n, A and L are as defined above,

L′₁ represents the substituent L₁ as defined above involved in acovalent bond with a functional group borne by a compound of interest{circle around (Y)}, said compound of interest {circle around (Y)} beingchosen from enzymes, nanoparticles, proteins, antibodies or cytotoxicagents.

According to one embodiment of the invention, the conjugate of formula(III) as defined above has an IC₅₀ value for the cation-independentmannose 6-phosphate receptor (CI-M6PR) of at least 10⁻⁵ M, andpreferably ranging from 10⁻⁶ to 10⁻⁹ M. The term “IC₅₀ value” isintended to mean the concentration of compounds capable of inhibitingthe bonding of CI-M6PR to its ligand, pentamannose 6-phosphate (PMP),adsorbed beforehand.

By way of more specific examples of the compound of interest {circlearound (Y)}, mention may be made of a lysosomal enzyme or ananoparticle, in particular a silica nanoparticle.

According to one embodiment of the invention, the nanoparticle mayincorporate a photosensibilizer of neutral porphyrin type.

By way of examples of lysosomal enzymes, mention may be made of thosechosen from acid alpha-glucosidase, acid beta-galactosidase-1, acidsphingomyelinase, alpha-D-mannosidase, alpha-fucosidase,alpha-galactosidase A, alpha-glucosaminide acetyltransferase,alpha-glucosidase, alpha-L-iduronidase, alpha-N-acetylgalactosaminidase,alpha-acetylglucosaminidase, alpha-D-neuraminidase, arylsulfatase A,arylsulfatase B, beta-galactosidase, beta-glucuronidase,beta-mannosidase, cathepsin D, cathepsin K, ceramidase, cystinosine,ganglioside activator GM2, galactocerebrosidase, glucocerebrosidase,heparan sulfatase, hexosaminidase A, hexosaminidase B, hyaluronidase,iduronate-2-sulfatase, LAMP2, lysosomal acid lipase,N-acetylglucosamine-1-phosphotransferase, N-acetylgalactosamine6-sulfatase, N-acetylglucosamine-1-phosphotransferase,N-acetylglucosamine-6-sulfate sulfatase,N-aspartyl-beta-glucosaminidase, palmitoyl-thioesterase-1, acidphosphatase, protected protein/cathepsin A (PPCA), sialin,tripeptidyl-peptidase 1.

Said enzymes are obtained by genetic engineering in production systemswhich make it possible to obtain biologically active and functionalrecombinant proteins. For example, for the production of lysosomalglycoproteins, the baculovirus/sf9 insect cells system is used.

The invention also relates to the use of at least one compound offormula (I) as defined above, for forming at least one covalent bondwith at least one functional group borne by a compound of interest{circle around (Y)}, said compound of interest {circle around (Y)} beingchosen from enzymes, nanoparticles, proteins, antibodies or cytotoxicagents.

According to one advantageous embodiment of the invention, n′ compoundsof formula (I) can react with n′ functional group(s) borne by saidcompound of interest Y, with n′ being an integer ranging from 1 to 1000,and preferably from 1 to 100, so as to form n′ covalent bonds.

The invention thus also relates to the use of n′ compound(s) of formula(I) for forming n′ covalent bond(s) with n′ functional group(s) of thecompound of interest {circle around (Y)}, with n′ as defined above.

A subject of the invention is also the use of n′ compound(s) of formula(I) as defined above, for forming the conjugate of general formula(IIIa):

in which P¹, X, n, n′, A, L, L′₁ and {circle around (Y)} are as definedabove.

When n′ is an integer equal to 1, then the conjugate of general formula(III) and the conjugate of general formula (IIIa) are identical.

The term “conjugate of general formula (III)” refers to a polysaccharideof formula (I) covalently bonded at its L₁ group with a functional groupof the compound of interest {circle around (Y)}.

The term “conjugate of general formula (IIIa)” refers, when n′ is notequal to 1, to at least two polysaccharide(s) of formula (I) covalentlybonded with at least two functional groups of the compound of interest{circle around (Y)}.

For example, when the compound of interest {circle around (Y)} is ananoparticle, the conjugate (IIIa) may comprise 100 polysaccharides offormula (I) forming respectively 100 covalent bonds with 100 functionalgroups borne by the nanoparticle.

The LL₁ group of the compounds (I) of the invention is as definedpreviously and is capable of binding by covalent bonding, directly orafter activation, to at least one of the functions naturally present onor artificially introduced onto the compound of interest {circle around(Y)}.

As previously mentioned through the definition of the substituents L andL₁, the substituent LL₁ of the compounds (I) of the invention may inparticular comprise a reactive group chosen from carboxylic acid andsalts thereof, acid chloride, ester (alkyl ester, p-nitrophenyl ester,succinimidyl ester, sulfosuccinimidyl ester, etc.), azido (acyl azide,azidonitrophenyl etc.), hydrazide, 3-acyl-1,3-thiazolidine-2-thione,substituted or unsubstituted amine, O-alkyloxyamine, quaternaryammonium, isocyanate, isothiocyanate, hydrazine, phthalimido, maleimide,haloacetamide, monochlorotriazine, dichlorotriazine, mono- ordi-halogenated pyridine, thiol, sulfonyl chloride, vinylsulfone,disulfide, hydroxyl, epoxide or imidazolyl.

In one particular embodiment, the substituent LL₁ comprises a reactivecarbonyl group chosen from acylhydrazide, hydrazine or O-alkyloxyamine.The reaction between said acylhydrazide, hydrazine or O-alkyloxyamineand a carbonyl group of the compound of interest {circle around (Y)}results respectively in an acylhydrazone bond, a hydrazone bond or anoxime bond. Chemical groups of this type are generally useful forbonding glycoproteins (compound of interest {circle around (Y)}): thecarbonyl groups (either naturally present or induced by oxidation ofhydroxyl functions of the glycosyl chains of the glycoprotein) availableon the fragments of oligosaccharide of the glycoprotein are reacted withthe reactive carbonyl groups of the compounds (I) of the invention.

By way of more particular examples:

-   -   carbonyl functions of the compound of interest {circle around        (Y)} can react with O-alkylamine or acylhydrazide functions of        the compound (I) (namely L₁=—O—NH₂ or —CO—NH—NH₂) in order to        result in the formation of oxime or acylhydrazone linkages;    -   amine functions of the compound of interest {circle around (Y)}        can react with ethyl squarate or activated ester functions of        the compound (I) (namely, for example,

or —COOH activated with N-hydroxysuccinimide (NHS) or withhydroxybenzotriazole (HOBt)) in order to result in the formation ofsquarate linkages or amide linkages;

-   -   thiol functions of the compound of interest {circle around (Y)}        can react with thiol, disulfide or maleimide functions of the        compound (I) (namely L₁=—SH, —S—S—R₁ with R₁ being C₁-C₅ alkyl,

in order to result in the formation of disulfide or thiolene linkages;

-   -   alkyne or azide functions of the compound of interest {circle        around (Y)} can react respectively with an azide function of the        compound (I) (namely L₁=N₃) or alkyne function (namely L₁=—C≡CH)        in order to result in the formation of a triazole (which        involves a cycloaddition reaction between the azide and the        alkyne).

According to the invention, the choice of the substituent LL₁ of thecompound (I) will depend on the nature of the compound of interest{circle around (Y)} and of the functional groups formed by the latter.

Indeed, those skilled in the art easily understand that the length ofthe substituent LL₁ will increase with the steric hindrance associatedwith the compound of interest {circle around (Y)} which must be bonded.

For example, if the compound of interest {circle around (Y)} is aprotein or glycoprotein, then a substituent LL₁ comprising no more than2 or 3 consecutive atoms will be sufficient to ensure a satisfactoryaffinity between the compound (I) and CI-M6PR.

On the other hand, if the compound of interest {circle around (Y)} is aprotein or a nanoparticle which causes considerable steric hindrance inproximity to the M6P-binding sites of CI-M6PR, then a substituent LL₁comprising more than 2 or 3 consecutive atoms will be required to ensurea satisfactory affinity between the compound (1) and CI-M6PR.

The present invention also relates to a process for preparing aconjugate of formula (III) as defined above or a conjugate of formula(IIIa) as defined above, characterized in that:

-   -   at least one functional group borne by a compound of interest        {circle around (Y)}, said compound of interest being as defined        above, is reacted with    -   at least one compound corresponding to the formula (I):

in which P¹, X, n, A, L and L₁ are as defined above.

According to one embodiment of the invention, a compound of formula (I)as defined above, in which P¹ represents a hydrogen, and X, n, A, L andL₁ are as defined above, will for example be reacted with at least onefunctional group borne by the compound of interest {circle around (Y)}as defined above.

Another subject of the invention relates to a conjugate of formula (III)as defined above or a conjugate of formula (IIIa) as defined above:

-   -   for use thereof in a method for therapeutic treatment of the        human or animal body, in particular chosen from enzyme        replacement therapy, photodynamic therapy or cancer treatment,        and/or    -   for use thereof in a method of diagnosis, in particular of        diagnosis of diseases or ailments associated with an increase or        decrease in CI-M6PR expression.

Indeed, by virtue of their high affinity for CI-M6PR, the conjugates(III) and (IIIa) of the invention are particularly useful for thediagnosis of diseases associated with CI-M6PR expression.

When the compound of interest {circle around (Y)} is a nanoparticleincorporating a photosensitizer of porphyrin type, then the conjugate ofthe invention of formula (III) or (IIIa) will be particularly suitablefor photodynamic therapy.

By way of examples of diseases that can be treated using the conjugates(III) or (IIIa) of the invention, mention may be made of diseases causedby a deficiency of the compound of interest {circle around (Y)} in thelysosome. This deficiency may be compensated for by the administrationof the conjugate of the invention, which is capable of specificallydelivering to the lysosome the compound of interest {circle around (Y)}that is deficient.

BRIEF DESCRIPTION TO THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The invention will be understood more clearly on reading the followingnonlimiting and purely illustrative examples, and the following FIGS. 1and 2.

FIG. 1 is a comparison of the cytotoxicity of the phosphonatedisaccharides 18, 19 (i.e. M6Pn-α(1,2)-Man-Ethyl-NH₂ andM6Pn-α(1,2)-Man-EthylSq) and carboxylate disaccharides 24 and 25 (i.e.M6C-α(1,2)-Man-Ethyl-NH₂ and M6C-α(1,2)-Man-EthylSq) of formula (I) ofthe invention on LNCaP cells and of their respective monosaccharidehomologs.

FIG. 2 represents the phototoxic effects, on LNCaP cells, of silicananoparticles alone (MSN) and of silica nanoparticles (MSN) grafted ontomonosaccharide carboxylate analogs (M6C-EthylSq) and disaccharidecarboxylate analogs 25 (M6C-α(1,2)-Man-EthylSq) at various incubationtimes. The nanoparticles grafted onto the monosaccharide carboxylateanalog and onto the disaccharide carboxylate analog are representedrespectively by “MSN-M6C-EthylSq” and “MSN-M6C-α(1,2)-Man-EthylSq”.

FIG. 3 indicates the assaying of the GAA catalytic activity in myoblastsof patients suffering from the adult form of Pomp disease afterincubation for 48 h in the presence of 50 nM rhGAA or of the conjugateof formula (III) of the invention, namely therhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino conjugate (28) (called rhGAA-M6Pn28in FIG. 3).

FIG. 4 presents the analysis of the amounts of mature GAA by Westernblot with an anti-human GAA antibody (anti-LYAG, Genetex) or ananti-human actin antibody (Invitrogen) after incubation of the myoblastsfor 48 h in the presence of 50 nM rhGAA or of the conjugate of formula(III) of the invention, namely the rhGAA-M6Pn-α(1,2)-Man-Ethyloxyaminoconjugate (28) (called rhGAA-M6Pn28 in FIG. 4).

EXAMPLE 1

Synthesis of Compounds of Formula (I)

This example describes the preparation of multi-functionalizeddisaccharides corresponding to the formula (I) in which n is an integerequal to 1; P¹ represents, independently of one another, H, (CH₃)₃Si— orC₆H₅CH₂—;

represents a single or double bond; X represents the phosphonate groupor the carboxylate group with Z representing, independently of oneanother, H, ethyl or Na; A represents oxygen; L represents —CH₂—CH₂—; L₁represents —Br, —NH₂, —N₃,

1) Preparation of Disaccharides 9, 10, 11 and 12 Corresponding to theFormula (II)

a) Preparation of a Saccharide Acceptor 6:3,4,6-tri-O-benzyl-α-D-mannopyranoside of 2-bromoethyl

The process of preparing a saccharide acceptor 6 corresponding to thegeneral formula (IVa), in which P¹ represents Bn, A represents O, Lrepresents CH₂—CH₂ and L₁ represents Br, is illustrated below:

The starting compound is pentaacetylated α-D-mannose onto which abromine is introduced in the anomeric position using 33% hydrobromicacid in acetic acid, so as to form the compound 1. The intermediate 1 isthen reacted with 2-bromoethanol and 2,6-lutidine so as to form theorthoester 2 with a yield of 63% over two steps. The acetates present onpositions 3, 4 and 6 of the orthoester 2 are then saponified so as togive the intermediate 3 with a yield of 82%. This deprotection step istotal and does not require purification. These same positions 3, 4 and 6are then benzylated through the action of benzyl bromide so as to formthe compound 4 with a yield of 56%. The orthoester 4 is then opened inthe presence of BF₃.Et₂O and 2-bromoethanol so as to form the keyintermediate 5 with a yield of 75%. The compound 5 is thusfunctionalized in the anomeric position and bears a protecting group inposition 2 orthogonal to the groups of positions 3, 4 and 6. It is thuspossible to selectively deprotect this position 2 using a sodium oxidesolution so as to obtain the intermediate 6 with a yield of 82%.

The saccharide acceptor 6 is functionalized in the anomeric position:the synthon 6 will therefore be common regardless of the desireddisaccharide of formula (II).

Conditions and reagents: (i) 33% HBr, AcOH, AT, 1 h; (ii)2-bromoethanol, 2,6-lutidine, DCM, 40° C., 3 h; (iii) 1N NaOH, THF, AT,16 h; (iv) BnBr, NaH, DMF, AT, 21 h; (v) 2-bromoethanol, BF₃.Et₂O, AT, 1h30; (vi) 1N NaOH, THF, AT, 21 h.

Experimental Section

Preparation of 1-bromo-2,3,4,6-tetra-Oacetyl-α-D-mannopyranose 1

10 g of α-D-mannose pentaacetate are dissolved in 20 ml of a solution ofhydrobromic acid at 33% in acetic acid. The yellow solution is stirredfor 1 h at ambient temperature. The reaction mixture is cooled to 0° C.,diluted with 30 ml of CH₂Cl₂ and neutralized using a saturated solutionof NaHCO₃. The aqueous phase is extracted with CH₂Cl₂ (5×100 ml). Theorganic phases are combined, dried over MgSO₄ and concentrated so as togive the compound 1 (11.9 g) which is used for the next step withoutpurification.C₁₄H₁₉BrO₉  Chemical formula 1:

Exact weight: 410.02 g·mol⁻¹

R_(f): 0.76 [EtOAc/Cyclo (1:1)]

Preparation of1,2-O-(1-(2-bromoethoxy)ethylidene)-3,4,6-tri-O-acetyl-α-D-mannopyranose2

11.08 g (1 eq; 26.9 mmol) of compound 1 are dissolved in 9.5 ml ofCH₂Cl₂ and 9.4 ml (3 eq; 80.7 mmol) of 2,6-lutidine. 4.8 ml (2.5 eq;67.25 mmol) of 2-bromoethanol are added. The orange-colored reactionmedium is heated to 40° C. and stirred for 4 h. A white precipitate isformed. The solution is cooled to ambient temperature and 12 ml of Et₂Oare added. The precipitate is filtered off. The filtrate is diluted with30 ml of CH₂Cl₂ and washed successively with 30 ml of water, 30 ml of asaturated solution of NaHCO₃ and 30 ml of brine. The combined aqueousphases are extracted with 50 ml of CH₂Cl₂. The organic phases arecombined and then dried over MgSO₄ and concentrated. The residueobtained is dissolved in 20 ml of CH₂Cl₂ and 25 ml of ethanol are added.The solution is cooled to −30° C. so as to give the compound 2 in theform of white crystals with a yield of 51% (6.268 g; 13.8 mmol) over twosteps.C₁₆H₂₃BrO₁₀  Chemical formula 2:

Exact weight: 454.05 g·mol⁻¹

R_(f): 0.49 [Cyclo/EtOAc (1:1)]

MS, ESI⁺ m/z: 477 [M+Na]⁺

Preparation of 1,2-O-(1-(2-bromoethoxy)ethylidene-α-D-mannopyranose 3

4.79 g (1 eq; 10.6 mmol) of compound 2 are dissolved in 22 ml of THF. 43ml of a 1N aqueous NaOH solution are added. The solution becomes cloudy.2 ml of methanol are added and the solution becomes clear. The reactionmixture is stirred at ambient temperature for 16 h. 250 ml of ethylacetate are added and the aqueous phase is extracted with 3×200 ml ofethyl acetate. The organic phases are combined and then dried over MgSO₄and concentrated. The residue obtained is purified by automated flashchromatography on silica gel (CH₂Cl₂/MeOH: from 0 to 5% in 15 min; 5%for 10 min; from 5 to 10% in 15 min) so as to give the compound 3 in theform of a colorless oil with a yield of 82% (2.86 g; 8.69 mmol).C₁₀H₁₇BrO₇  Chemical formula 3:

Exact weight: 328.02 g·mol⁻¹

R_(f): 0.36 [CH₂Cl₂/MeOH (9:1)]

MS, ESI⁻ m/z: 373 [M−H+HCOOH]⁻

Preparation of1,2-O-(1-(2-bromoethoxy)ethylidene-3,4,6-tri-O-benzyl-α-D-mannopyranose4

2.86 g (1 eq; 8.69 mmol) of the compound 3 are dissolved in 40 ml ofDMF. 4.2 ml (4 eq; 34.76 mmol) of benzyl bromide are added. The reactionmixture is cooled to 0° C. and 874 mg (3 eq; 26 mmol) of a 60%dispersion of NaH in oil are added. After stirring for 17 h30 at ambienttemperature, partially benzylated product remains. The addition of oneequivalent of benzyl bromide and of NaH does not cause the reaction toprogress. 50 ml of Et₂O and 40 ml of water are added. The aqueous phaseis extracted with 2×100 ml of Et₂O. The organic phases are combined,washed with 5×80 ml of water and then dried over MgSO₄ and concentrated.The residue obtained is purified by automated flash chromatography onsilica gel (cyclohexane/Et₂O: from 0 to 10% in 15 min; 10% for 10 min;from 10 to 20% in 15 min; 20% for 50 min; from 20 to 40% in 30 min) soas to give the compound 4 in the form of a white solid with a yield of56% (2.92 g; 4.87 mmol).C₃₁H₃₅BrO₇  Chemical formula 4:

Exact weight: 598.16 g·mol⁻¹

R_(f): 0.43 [Cyclo/Et₂O (1:1)]

MS, ESI⁺ m/z: 621 [M+Na]⁺

Preparation of 2-bromoethyl2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranoside 5

1 g (1 eq; 1.67 mmol) of the compound 4 is dissolved of 20 ml of CH₂Cl₂.236 μl (2 eq; 3.34 mmol) of 2-bromoethanol are added and the reactionmixture is stirred at ambient temperature for 10 min. 211 μl (1 eq; 1.67mmol) of BF₃.Et₂O. The reaction mixture is stirred at ambienttemperature for 16 h. The solution became pale yellow. The reaction isdiluted with 50 ml of saturated solution of NaHCO₃. The aqueous phase isextracted with 3×50 ml of CH₂Cl₂. The organic phases are combined,washed with 100 ml of brine and then dried over MgSO₄ and concentrated.The residue obtained is purified by automated flash chromatography onsilica gel (cyclohexane/Et₂O: 0 to 30% in 30 min; 30% for 20 min) so asto give the compound 5 in the form of a colorless oil with a yield of75% (750 mg; 1.25 mmol).C₃₁H₃₅BrO₇  Chemical formula 5:

Exact weight: 598.16 g·mol⁻¹

R_(f): 0.45 [Cyclo/Et₂O (1:1)]

MS, ESI⁺ m/z: 621 [M+Na]⁺

Preparation of 2-bromoethyl 3,4,6-tri-O-benzyl-α-D-mannopyranoside 6

750 mg (1 eq; 1.25 mmol) of the compound 5 are dissolved in 1.6 ml ofTHF. 2.5 ml (2 eq; 2.5 mmol) of a 1N NaOH solution are added. Thesolution becomes cloudy. The solution is stirred at ambient temperaturefor 21 h. The solution is neutralized with a 1N HCl solution. 40 ml ofCH₂Cl₂ are added. The aqueous phase is extracted with 3×40 ml of CH₂Cl₂.The organic phases are combined and then dried over MgSO₄ andconcentrated. The residue obtained is purified by automated flashchromatography on silica gel (cyclohexane/Et₂O: from 0 to 20% in 15 min;20% for 10 min; from 20 to 30% in 15 min; 30% for 15 min) so as to givethe compound 6 in the form of a colorless oil with a yield of 82% (575mg; 1.03 mmol).C₂₉H₃₃BrO₆  Chemical formula 6:

Exact weight: 556.16 g·mol⁻¹

R_(f): 0.29 [Cyclo/Et₂O (7:3)]

MS, ESI⁺ m/z: 579 [M+Na]⁺

b) Preparation of a Saccharide Donor 8:2,3,4,6-tetra-O-acetyl-α-D-mannopyranose trichloroacetimidate

The process for preparing a saccharide donor compound 8 corresponding tothe general formula (V), in which P¹ represents Ac, is illustratedbelow:

The starting compound is pentaacetylated α-D-mannose which isdeprotected in the anomeric position using morpholine so as to give thecompound 7 without purification. The trichloroacetimidate 8 is thenformed by reacting the compound 7 with trichloroacetonitrile in thepresence of DBU.

Conditions and reagents: (i) morpholine, DCM, reflux, 3 h30; (ii)Cl₃CCN, DBU, DCM, AT, 4 h.

Experimental Section

Preparation of 2,3,4,6-tetra-O-acetyl-D-mannose 7

3 g (1 eq; 7.69 mmol) of α-D-mannose pentaacetate are dissolved in 18 mlof CH₂Cl₂. 2.7 ml (4 eq; 30.76 mmol) of morpholine are added. Thereaction mixture is refluxed for 3 h30 and then cooled to ambienttemperature. The solution is neutralized with a 1N HCl solution. Theorganic phase is washed with 3×10 ml of water, dried over MgSO₄ andconcentrated so as give the product 7 in the form of a yellow oil thatis directly used for the next step.C₁₄H₂₀O₁₀  Chemical formula 7:

Exact weight: 348.30 g·mol⁻¹

R_(f): 0.38 [Cyclo/EtOAc (1:1)]

MS, ESI⁻ m/z: 393 [M−H+HCOOH]⁻

Preparation of trichloroacetimidate2,3,4,6-tetra-O-acetyl-α-D-mannopyranose 8

1.34 g (1 eq; 3.85 mmol) of the compound 7 are dissolved in 8.6 ml ofCH₂Cl₂. The yellow-colored solution is cooled to 0° C. 2.7 ml (7 eq;26.95 mmol) of trichloroacetonitrile and 575 μl of DBU(1.8-diazabicyclo[5.5.0]undec-7-ene) are added. The solution becomesbrown. The reaction medium is stirred for 4 h at ambient temperature andthen concentrated. The residue obtained is purified by automated flashchromatography on silica gel (cyclohexane/Et₂O: 50% for 25 min) so as togive the compound 8 in the form of a pale yellow oil with a yield of 45%(860 mg; 1.75 mmol). The relatively unstable product is rapidly used.C₁₆H₂₀Cl₃NO₁₀  Chemical formula 8:

Exact weight: 492.69 g·mol⁻¹

R_(f): 0.4 [Cyclo/Et₂O (3:7)]

c) Preparation of the Disaccharides 9, 10, 11 and 12

The process for preparing disaccharides 9, 10, 11 and 12 correspondingto the formula (II) is illustrated below:

The glycosylation reaction between the acceptor of formula IV or IVa, inparticular the saccharide 6, and the donor of formula V, in particularthe saccharide 8, makes it possible to obtain the compound of formula(II), in particular the disaccharide 12, which occupies a centralposition in the synthesis strategy of the invention. This is because thecompound of formula (II) makes it possible to diverge toward all of thecompounds of formula (I) of the invention. The isosteric analogs of M6Pof the invention (I) are in fact accessible starting from the compoundof formula (II).

The synthesis process of the invention imparts flexibility in theproduction of the disaccharide analogs of formula (I) of the invention,but also to the tri- or tetrasaccharide analogs of formula (I) of theinvention.

The respective “saccharide acceptor” 6 and “saccharide donor” 8compounds as prepared above are reacted in the presence oftrimethylsilyl triflate so as to form the disaccharide 9 with a yield of60%. The acetate groups are then saponified so as to give the compound10 with a yield of 74%. The alcohols thus being protected are thentrimethylsilylated through the action of trimethylsilyl chloride so asto form the intermediate 11. Position 6 is then selectively desilylatedin the presence of potassium carbonate in methanol so as to give thedisaccharide 12 with a yield of 81% over two steps.

Conditions and reagents: (i) TMSOTf, DCM, −30° C., 30 min; (ii) 1N NaOH,THF, AT, 18 h; (iii) TMSCl, NEt₃, DCM, AT, 18 h; (iv) K₂CO₃, MeOH, AT, 1h45.

Experimental Section

Preparation of bromoethyl2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside9

430 mg (1 eq; 0.77 mmol) of the compound 6 and 860 mg (2.27 eq; 1.75mmol) of the compound 8 are dissolved in 15 ml of CH₂Cl₂ in the presenceof 7 g of pre-activated molecular sieve 4 Å. The reaction medium isstirred for 30 min at ambient temperature and then cooled to −30° C. 167μl (1.2 eq; 0.924 mmol) of TMSOTf are added dropwise. The reactionmixture is stirred at −30° C. for 1 h15. The reaction medium is thenneutralized with 3.5 ml of pyridine. After filtration through celite,the filtrate is concentrated and then co-evaporated with toluene. Theresidue (pale yellow solid) is purified by automated flashchromatography on silica gel (cyclohexane/Et₂O: 30% for 110 min) so asto give the product 9 in the form of a colorless oil with a yield of 64%(441 mg; 0.497 mmol).C₄₃H₅₁BrO₁₅  Chemical formula 9:

Exact weight: 886.24 g·mol⁻¹

R_(f): 0.56 [Cyclo/Et₂O (2:8)]

MS, ESI⁺ m/z: 909 [M+Na]⁺

Preparation of bromoethylα-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside 10

5.84 g (1 eq, 6.58 mmol) of the compound 9 are dissolved in 16 ml ofTHF. 33 ml (5 eq, 32.9 mmol) of a 1 M sodium hydroxide solution areadded. The solution becomes cloudy. 1.5 ml of methanol are added. Thesolution is stirred at ambient temperature for 18 h and then neutralizedwith a 1M HCl solution and concentrated. The residue obtained ispurified by flash chromatography on silica gel (DCM/MeOH: 0% (500 ml);5% (500 ml); 7% (500 ml); 10% (1 L)) so as to give the compound 10 in aform of a white foam with a yield of 74% (3.5 g, 4.86 mmol).C₃₅H₄₃BrO₁₁  Chemical formula 10:

Molar mass: 718.2 g·mol⁻¹

R_(f): 0.41 [DCM/MeOH (9:1)]

Preparation of bromoethyl2,3,4,6-tetra-O-trimethylsilyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside11

3.4 g (1 eq, 4.72 mmol) of the compound 10 are dissolved in 24 ml offreshly distilled DCM and 19 ml (30 eq; 141.6 mmol) of triethylamine.The solution is cooled to 0° C. and 4.8 ml (8 eq; 37.8 mmol) oftrimethylsilyl chloride are added dropwise. After stirring for 18 h atambient temperature, a small amount of starting product remains. 1.2 ml(2 eq; 9.44 mmol) of trimethylsilyl chloride are added dropwise. Afterstirring for 1 h30 at ambient temperature, there is no progression. Thesolution is concentrated. The pink residue is dissolved in cyclohexaneand filtered through celite. The filtrate is concentrated so as toobtain the compound 11 in the form of a light brown oil that is directlyused for the next step.C₄₇H₇₅BrO₁₁Si₄  Chemical formula 11:

Molar mass: 1008.34 g·mol⁻¹

R_(f): 0.78 [Cyclo/Et₂O (7:3)]

Preparation of bromoethyl2,3,4-tri-O-trimethylsilyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside12

4.58 g (4.72 mmol; 1 eq) of the compound 11 are dissolved in 65 ml offreshly distilled methanol. A solution of 6.5 mg (0.01 eq; 0.0472 mmol)of K₂CO₃ (final concentration=0.63 mM) in 10 ml of methanol is addeddropwise. The reaction medium is stirred for 1 h45 at ambienttemperature and then diluted with 200 ml of CH₂Cl₂ and washed with 125ml of brine. The aqueous phase is extracted with 260 ml of CH₂Cl₂. Theorganic phases are combined, dried over MgSO₄ and concentrated. Theresidue obtained (light brown oil) is purified by chromatography onsilica gel (Cyclohexane/Et₂O: 30% (700 ml); 40% (500 ml); 50% (500 ml);70% (500 ml)) so as to give the compound 12 (3.58 g; 3.82 mmol) in theform of a colorless liquid with a yield of 81% over two steps.C₄₄H₆₇BrO₁₁Si₃  Chemical formula 12:

Molar mass: 936.16 g·mol⁻¹

R_(f): 0.45 [Cyclo/Et₂O (1:1)]

MS, ESI⁺ m/z: 959 [M+Na]⁺

2) Preparation of Disaccharides 14, 15, 16, 17, 18 and 19 Correspondingto the Formula (I) in which X Represents the Phosphonate Group

The process for preparing disaccharides 14, 15, 16, 17, 18 and 19corresponding to the formula (I) in which X represents the phosphonategroup is illustrated below:

The alcohol 12 obtained as described previously is oxidized to aldehydeusing Dess-Martin periodinane so as to form the compound 13. This keyintermediate will allow access to the various disaccharides of formula(I).

In order to synthesize the “phosphonate” disaccharides of formula (I),the aldehyde 13 is reacted with tetraethyl methylenediphosphonate,deprotonated beforehand with sodium hydride, so as to form compound 14with a yield of 75% over two steps. During a catalytic hydrogenationstep, in the presence of palladium-on-carbon and for which the hydrogenis generated in situ by gradual addition of triethylsilane, the doublebond and also the three benzyls are reduced and the trimethylsilylgroups are hydrolyzed. The compound 15 is thus obtained, withoutpurification, with a quantitative yield. The bromine atom is thenreplaced with potassium phthalimide so as to form the intermediate 16with a yield of 75%. The alcohol functions are then protected withtrimethylsilyl chloride so as to give the compound 17 with a yield of88%. The intermediate 17 thus obtained, without purification, is thenreacted with trimethylsilyl chloride and sodium iodide so as to formbis(trimethylsilyl) phosphonate by a Rabinowitz type reaction. Thisintermediate is then converted to phosphonic acid through the action ofhydrazine monohydrate in methanol, which will also displace thetrimethylsilyl groups present on the secondary alcohols of the sugar andreact with the phthalimide in order to mask the amine function. Thecompound 18 deprotected on the phosphonate part, on the alcohols of thetwo sugars, and bearing the amine function in the anomeric position isthus obtained, in a single step, with a yield of 76% over two steps. Thecompound 18 is then reacted with diethyl squarate so as to form theproduct 19 with a yield of 59%.

Conditions and reagents: (i) Dess-Martin periodinane, DCM, AT, 4 h (ii)tetraethyl methylenediphosphonate, NaH, THF, AT, 45 min; (iii) Pd/C,Et₃SiH, MeOH; (iv) potassium phthalimide, DMF, 60° C., 40 h; (v) TMSCl,NEt₃, DCM, DMF, AT, 24 h; (vi)-a TMSCl, NaI, ACN, 35° C., 1 h; (vi)-bN₂H₄.H₂O, MeOH, AT, 1 h30; (vii) diethyl squarate, EtOH/H₂O, NEt₃.

Experimental Section

Preparation of bromoethyl2,3,4-tri-O-trimethylsilyl-α-D-manno-hexodialdopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside13

1 g (1 eq; 1.07 mmol) of the compound 12 is dissolved in 24 ml of DCM onmolecular sieve. 5.4 ml (1.5 eq; 1.6 mmol) of a solution of Dess-Martinperiodinane at 0.3 M in dichloromethane are added dropwise. The reactionmedium is stirred at ambient temperature for 4 h and then diluted with120 ml of Et₂O. 25 ml of a saturated solution of NaHCO₃ and 3.9 g ofNa₂S₂O₃ are added. The solution is stirred at ambient temperature for 5min. The aqueous phase is extracted with 3×125 ml of Et₂O. The organicphases are combined, dried over MgSO₄ and concentrated so as to give thecompound 13 that is used directly for the next step.C₄₄H₆₅BrO₁₁Si₃  Chemical formula 13:

Molar mass: 934.14 g·mol⁻¹

R_(f): 0.77 [DCM/Et₂O (95:5)]

Preparation of bromoethyl2,3,4-tri-O-trimethylsilyl-6,7-dideoxy-7-diethoxyphosphinyl-αD-manno-hept-6-enopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyranoside14

90 mg (2.5 eq; 2.67 mmol) of NaH as a 60% dispersion in oil aredissolved in 16 ml of THF. 530 μl (2 eq; 2.14 mmol) of tetraethylmethylenediphosphonate are added dropwise. The reaction mixture isstirred for 45 min at ambient temperature and then added to 998 mg (1eq; 1.07 mmol) of the compound 13 dissolved in 8 ml of THF. The solutionturns yellow in color and then becomes brown. The reaction medium isstirred at ambient temperature for 45 min and then diluted with 200 mlof CH₂Cl₂ and washed with 2×40 ml of brine. The aqueous phase isextracted with 3×130 ml of CH₂Cl₂. The organic phases are combined,dried over MgSO₄ and concentrated. The residue obtained (brown oil) ispurified by automated flash chromatography on silica gel (DCM/Et₂O: 0%to 5% in 15 min; 5% for 10 min; from 5% to 10% in 15 min; 10% for 10min; from 10 to 13% in 15 min) so as to give the compound 14 in the formof a light yellow oil with a yield of 76% (864 mg; 0.81 mmol) over twosteps.C₄₉H₇₆BrO₁₃Si₃  Chemical formula 14:

Molar mass: 1068.25 g·mol⁻¹

R_(f): 0.26 [DCM/Et₂O (95:5)]

Preparation of bromoethyl6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside15

864 mg (0.81 mmol) of the compound 14 are dissolved in 8.2 ml ofmethanol. 130 mg (15% by weight) of palladium-on-carbon are added. 1.3ml (8.1 mmol; 10 eq) of triethylsilane are added dropwise for 1 h20. Thesuspension is stirred at ambient temperature for 30 min and thenfiltered through celite. The filtrate is evaporated so as to give thecompound 15 with a yield of 99% (468 mg; 8.0 mmol).C₁₉H₃₆BrO₁₃P  Chemical formula 15:

Exact weight: 582.2 g·mol⁻¹

R_(f): 0.23 [DCM/MeOH (85:15)]

MS, ESI⁺ m/z: 583.1 [M+H]⁺

Preparation of phthalimidoethyl6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside16

220 mg (0.38 mmol) of the compound 15 are dissolved in 1.8 ml of DMF onmolecular sieve. 119 mg (0.64 mmol; 1.7 eq) of potassium phthalimide areadded. The suspension is stirred for 40 h at 60° C. and then cooled toambient temperature and concentrated. The residue obtained is purifiedby chromatography on silica gel (EtOAc/MeOH: 75/25) so as to give thecompound 16 in the form of a colorless solid with a yield of 75% (184mg; 0.28 mmol).C₂₇H₄₀NO₁₅P  Chemical formula 16:

Molar mass: 649.58 g·mol⁻¹

R_(f): 0.31 [EtOAc/MeOH (75:25)]

MS, ESI⁺ m/z: 650.3 [M+H]⁺

Preparation of phthalimidoethyl2,3,4-tri-O-trimethylsilyl-6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-trimethylsilyl-α-D-mannopyranoside17

184 mg (0.28 mmol) of the compound 16 are dissolved in 817 μl of freshlydistilled DCM and 1.15 ml (8.5 mmol; 30 eq) of triethylamine. Thestarting product is not completely soluble. 431 μl (3.4 mmol; 12 eq) oftrimethylsilyl chloride are added dropwise. The solution is stirred atambient temperature for 15 h. The starting product is still notcompletely dissolved and the solution is white in color. 0.5 ml of DMFon molecular sieve are added, the starting product is then soluble. Thesolution is stirred at ambient temperature for 24 h and then evaporated.During the reaction, the solution becomes brown. The brown residue isdissolved in cyclohexane and then filtered through celite. The filtrateis concentrated so as to give the compound 17 with a yield of 88% (268mg; 0.25 mmol) which is directly used for the next step.C₄₅H₈₈NO₁₅PSi₆  Chemical formula 17:

Molar mass: 1082.66 g·mol⁻¹

R_(f): 0.49 [DCM/Et₂O (9:1)]

Preparation of aminoethyl6-deoxy-6-dihydroxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-D-mannopyranoside(M6Pn-α(1,2)-Man-Ethyl-NH₂) 18

269 mg (0.25 mmol) of the compound 17 and 260 mg (1.73 mmol; 7 eq) ofsodium iodide are dissolved in 7.3 ml of freshly distilled acetonitrile.219 μl (1.73 mmol; 7 eq) of trimethylsilyl chloride are added dropwise.The solution is stirred at 35° C. for 1 h15. A precipitate forms duringthe reaction. The supernatant is drawn off with a tube into around-bottomed flask and evaporated to dryness. 132 μl (2.72 mmol; 11eq) of hydrazine monohydrate are diluted in 3.4 ml of methanol onmolecular sieve and added to the previously obtained residue. A whiteprecipitate is formed. The suspension is stirred at ambient temperaturefor 1 h30. The precipitate is dissolved in water and evaporated. Theresidue obtained is purified by chromatography on silica gel(isopropanol/aqueous ammonia/water: 5/3/2) so as to give the compound 18in the form of a white solid with a yield of 76% (67 mg; 0.14 mmol).C₁₅H₃₀NO₁₃P  Chemical formula 18:

Molar mass: 463.37 g·mol⁻¹

R_(f): 0.49 [DCM/Et₂O (9:1)]

HRMS: mass calculated: 464.1533; mass found: 464.1530.

Preparation of (4-ethoxy-2,3-dioxocyclobut-1-enyl)aminoethyl6,7-dideoxy-7-dihydroxyphosphinyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside(M6Pn-α(1,2)-Man-EthylSq) 19

The compound 18 (32 mg; 1 eq; 0.07 mmol) is dissolved in anethanol/water mixture (1:1) (620 μl). Diethyl squarate (10.3 μl; 1 eq;0.07 mmol) and triethylamine (9.7 μl; 1 eq; 0.07 mmol) are added. Thereaction is stirred at ambient temperature for 20 h and the solvent isthen evaporated off. The residue is precipitated from an EtOAc/MeOHmixture (7:3). After centrifugation, the precipitate is rinsed fivetimes with ethyl acetate so as to give the compound 19 with a yield of59% (24 mg; 0.041 mmol).C₂₁H₃₄NO₁₆P  Chemical formula 19:

Molar mass: 587.47 g·mol⁻¹

R_(f): 0.67 [MeOH/H₂O (9:1)]

MS, ESI m/z: 586 [M−H]⁻

3) Preparation of Disaccharides 20, 21, 22, 23, 24, and 25 Correspondingto the Formula (I) in which X Represents the Carboxylate Group

The process for preparing disaccharides 20, 21, 22, 23, 24 and 25corresponding to the formula (I) in which X represents the carboxylategroup is illustrated below:

The beginning of the synthesis of the “carboxylate” disaccharides offormula (I) is identical to that of the “phosphonate” disaccharides,namely the aldehyde 13 is obtained from the alcohol 12 in the same wayas described above.

The aldehyde 13 is then reacted with triethyl phosphonoacetatedeprotonated beforehand using sodium hydride so as to form the compound20 with a yield of 75% over two steps. The double bond and also thethree benzyl groups are reduced in a single hydrogenation step. Duringthis reaction, the trimethylsilyl groups are also hydrolyzed. Thisreaction is carried out using palladium-on-carbon at 10% and thehydrogen is generated in situ by gradual addition of triethylsilane soas to form the compound 21 with a yield of 95%. This hydrogenation stepis total and does not require purification in order to obtain the purecompound 21. The bromine atom is then substituted with sodium azide soas to give the compound 22 with a yield of 99%. The ester function isthen saponified so as to form the compound 23 with a yield of 86%.Finally, the azide function is reduced during a catalytic hydrogenationreaction using palladium-on-carbon and triethylsilane so as to form thecompound 24 with a yield of 97%. Finally, the compound 24 is reactedwith diethyl squarate so as to give the compound 25 with a yield of 73%.

Conditions and reagents: (i) Dess-Martin periodinane, DCM, AT, 4 h; (ii)triethyl phosphonoacetate, NaH, THF, AT, 14 h; (iii) Pd/C, Et₃SiH, MeOH,AT; (iv) NaN₃, DMF, AT, 5d; (v) 1N NaOH, AT, 20 h; (vi) Pd/C, Et₃SiH,MeOH/H₂O; (vii) diethyl squarate, EtOH/H₂O, NEt₃, AT, 2 h30.

Experimental Section

Preparation of bromoethyl2,3,4-tri-O-trimethylsilyl-6,7-dideoxy-7-ethoxycarbonyl-α-D-manno-hept-6-enopyranosyl-(1→2)-3,4,6-tri-O-benzyl-α-D-mannopyrannoside20

144 mg (4 eq; 4.28 mmol) of NaH 60% dispersion in oil are dissolved in10 ml of THF. 637 μl (3 eq; 3.21 mmol) of triethyl phosphonoacetate areadded dropwise. The reaction mixture is stirred for 45 min at ambienttemperature and then added to 1.065 g (1 eq; 1.07 mmol) of the compound13 dissolved in 20 ml of THF. The solution turns orange. The reactionmedium is stirred at ambient temperature for 14 h30 and then dilutedwith 175 ml of CH₂Cl₂ and washed with 4×50 ml of brine. The aqueousphase is extracted with 125 ml of CH₂Cl₂. The organic phases arecombined, dried over MgSO₄ and concentrated. The residue obtained(orange oil) is purified by automated flash chromatography on silica gel(Cyclohexane/Et₂O: 20% (900 ml)) so as to give the compound 20 in theform of a colorless oil with a yield of 75% (802 mg; 0.80 mmol) over twosteps.C₄₈H₇₁BrO₁₂Si₃  Chemical formula 20:

Molar mass: 1004.23 g·mol⁻¹

R_(f): 0.39 [Cyclo/Et₂O (8:2)]

Preparation of bromoethyl6,7-dideoxy-7-ethoxycarbonyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside21

795 mg (1 eq; 0.79 mmol) of the compound 20 are dissolved in 8 ml ofmethanol. 160 mg (20% by weight) of palladium-on-carbon at 10% areadded. 1.3 ml (10 eq; 7.9 mmol) of triethylsilane are added dropwise for1 h. The reaction medium is stirred at ambient temperature for 20 minand then filtered through celite (rinsed with hot methanol). Thefiltrate is concentrated so as to give the compound 21 in the form of acolorless oil with a yield of 95% (390 mg; 0.75 mmol).C₁₈H₃₁BrO₁₂  Chemical formula 21:

Molar mass: 519.34 g·mol⁻¹

R_(f): 0.40 [DCM/MeOH (85:15)]

Preparation of azidoethyl6,7-dideoxy-7-ethoxycarbonyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside22

440 mg (1 eq; 0.85 mmol) of the compound 21 are dissolved in 3.5 ml offreshly distilled DMF. 66 mg (1.2 eq; 1.02 mmol) of sodium azide areadded. The solution is stirred at ambient temperature for five days andthen evaporated. The residue obtained is purified by flashchromatography on silica gel (EtOAc/MeOH: 10% (210 ml); 20%) so as togive the compound 22 in the form of a white solid with a yield of 99%(407 mg; 0.85 mmol).C₁₈H₃₁N₃O₁₂  Chemical formula 22:

Molar mass: 481.45 g·mol⁻¹

R_(f): 0.43 [EtOAc/MeOH (8:2)]

MS, ESI⁺ m/z: 482 [M+H]⁺

Preparation of azidoethyl6,7-dideoxy-7-hydroxycarbonyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside23

377 mg (1 eq; 0.78 mmol) of the compound 22 are dissolved in 932 μl (1.2eq; 0.932 mmol) of a 1 N sodium hydroxide solution. The solution isstirred at ambient temperature for 20 h and then concentrated. Theresidue obtained is purified by flash chromatography on silica gel(isopropanol/35% aqueous ammonia/water: 6/3/1) so as to give thecompound 23 in the form of a white solid with a yield of 86% (316 mg;0.67 mmol).C₁₆H₂₇N₃O₁₂  Chemical formula 23:

Molar mass: 453.40 g·mol⁻¹

R_(f): 0.38 [isopropanol/NH₄OH/water (6:3:1)]

MS, ESI⁺ m/z: 454 [M−H]⁺

Preparation of aminoethyl6,7-dideoxy-7-hydroxycarbonyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside(M6C-α(1,2)-Man-Ethyl-NH₂) 24

316 mg (1 eq; 0.67 mmol) of the compound 23 are dissolved in 15 ml of a2:1 methanol/water mixture. 32 mg (10% by weight) of palladium-on-carbonat 10% are added. 536 μl (5 eq; 3.36 mmol) of triethylsilane are addeddropwise for 40 min. After stirring for 45 min at ambient temperature,the starting product is still present. 536 μl (5 eq; 3.36 mmol) oftriethylsilane are added dropwise for 30 min. The reaction medium isstirred at ambient temperature for 10 min (no change) and then filteredthrough celite (rinsed with hot methanol). The filtrate is concentratedand then dissolved in 3 ml of a 2:1 methanol/water mixture. 60 mg (20%by weight) of palladium-on-carbon at 10% are added. 1.07 ml (10 eq; 6.72mmol) of triethylsilane are added dropwise for 20 min. The suspension isstirred at ambient temperature for 30 min and then filtered throughcelite (rinsed with hot methanol). The filtrate is concentrated so as togive the compound 24 in the form of a colorless oil with a yield of 97%(280 mg; 0.66 mmol).C₁₆H₂₉NO₁₂  Chemical formula 24:

Molar mass: 427.40 g·mol⁻¹

R_(f): 0.51 [isopropanol/NH₄OH/water (5:4:1)]

MS, ESI⁺ m/z: 428 [M+H]⁺

Preparation of (4-ethoxy-2,3-dioxocyclobut-1-enyl)aminoethyl6,7-dideoxy-7-hydroxycarbonyl-α-D-manno-heptopyranosyl-(1→2)-α-D-mannopyranoside(M6C-α(1,2)-Man-EthylSq) 25

45 μl (1 eq; 0.307 mmol) of diethyl squarate are diluted in 300 μl of a2:1 ethanol/water mixture. The pH of the solution is 5. 20 μl (0.5 eq;0.154 mmol) of triethylamine are added so as to achieve a pH of 8-9. 131mg (1 eq; 0.307 mmol) of the compound 24 dissolved in 1.7 ml of a 2:1ethanol/water mixture are added dropwise. The solution is stirred atambient temperature for 2 h30, while maintaining the pH at 8-9 by addingtriethylamine, and is then concentrated. The residue obtained ispurified by flash chromatography on silica gel (EtOAc/MeOH: deposition3:7; elution: 30% (20 ml); 40% (10 ml); 50% (20 ml); 70% (30 ml) so asto give the compound 25 in the form of a colorless solid with a yield of73% (123 mg; 0.223 mmol).C₂₂H₃₃NO₁₅  Chemical formula 25:

Molar mass: 551.50 g·mol⁻¹

R_(f): 0.36 [EtOAc/MeOH (1:1)]

MS, ESI⁺ m/z: 552 [M+H]⁺

EXAMPLE 2

Study of the Disaccharide Analogs 18, 19, 24 and 25 of the Invention,Corresponding to the General Formula (I)

1) Binding of the M6P Analogs of the Invention with CI-M6PR

96-Well plates (Maxisorp Nunc) are incubated overnight at 4° C. with 200μl of PMP (pentamannose 6-phosphate) at the concentration of 200μg·ml⁻¹, in carbonate buffer (NaHCO₃/Na₂CO₃ at 0.1M, pH 9.6). Thefollowing day, the solution containing the residual PMP is discarded andthe wells are saturated, for 1 h at ambient temperature, with 360 μl of1% gelatin (Type A from Porcine Skin) diluted in PBS (1.9 mM NaH₂PO₄,8.1 mM Na₂PO₄ and 154 mM NaCl, pH 7.4). The wells are then rinsed fivetimes with PBS to which 0.2% gelatin has been added. All the washes andalso the dilutions are carried out in the solution of PBS to which 0.2%gelatin has been added. The M6P analogs to be tested at the variousconcentrations (from 10⁻² to 10⁻⁷ M) are pre-incubated in the presenceof pre-biotinylated CI-M6PR (M6PRb) (2.5 μg·mol⁻¹) for 20 min, then 200μl of the mixture are incubated in the wells for 2 h, at ambienttemperature. After three washes, the wells are incubated for 1 h with astreptavidin-peroxidase solution (250 μl per well; 3.10⁻⁸ M). Afterthree more washes, 200 μl of a solution of OPD (o-phenylenediamine, 1mg·ml⁻¹ in citrate buffer, pH 5.0, and 1 μl 30% H₂O₂.ml⁻¹; SigmaAldrich) are added. After incubation for 20 min in the dark at ambienttemperature, the optical densities are measured at the wavelength of 450nm.

The affinities of the phosphonate disaccharide 18 and carboxylatedisaccharide 24 of formula (I) of the invention were measured andcompared with those of their respective monosaccharide homologs.

The affinity denotes the binding capacity (in the case in point by meansof a covalent bond) of the M6P analogs for CI-M6PR.

The relative affinity makes it possible to compare the affinity of themolecules with M6C-Ethyl-NH₂ taken as reference and to which a valueequal to 1 is assigned.

The results obtained are summarized in table 1 below.

The affinity of the phosphonate disaccharide 18 (row 1 of table 1),aminoethyl6-deoxy-6-dihydroxyphosphinylmethylene-α-D-mannopyranose-α-(1,2)-D-mannopyrannose,represented by M6Pn-α(1,2)-Man-Ethyl-NH₂, is thus compared with that ofthe phosphonate monosaccharide (row 2), represented M6Pn-Ethyl-NH₂.

The affinity of the carboxylate disaccharide 24 (row 3), aminoethyl6,7-dideoxy-7-hydroxycarbonyl-α-D-manno-hept-6-anopyranose-α-(1,2)-α-D-mannopyrannose,represented by M6C-α(1,2)-Man-Ethyl-NH₂, is thus compared with that ofthe carboxylate monosaccharide (row 4), represented M6C-Ethyl-NH₂.

TABLE 1 Relative M6P analogs Chemical structure IC₅₀ in M affinityCompound 18 of the invention M6Pn-α(1,2)-Man-Ethyl-NH₂

0.5 × 10⁻⁵M 19.8 M6Pn-Ethyl-NH₂

2.9 × 10⁻⁵M  3.4 Compound 24 of the invention M6C-α(1,2)-Man-Ethyl-NH₂

1.7 × 10⁻⁵M  5.8 M6C-Ethyl-NH₂

9.9 × 10⁻⁵M  1  

Conclusion:

A better affinity of the disaccharide analogs 18 and 24 of the inventionis observed for CI-M6PR than that of the same analogs that aremonosaccharide analogs. Furthermore, a gain in affinity of thephosphonate analog 18 is observed compared with the carboxylate analog24, both in the disaccharide series and in the monosaccharide series.

2) Evaluation of the Cytotoxicity of the M6P Analogs of the Inventionwith CI-M6PR

The cytotoxicity of the phosphonate disaccharide analogs 18, 19 andcarboxylate disaccharide analogs 24, 25 of the invention was evaluatedon LNCaP prostate cancer cells, and was compared with that of theirrespective monosaccharide homologs.

The disaccharide analogs 18, 19, 24 and 25 of formula (I) of theinvention and also the monosaccharide analogs of the disaccharideanalogs 19, 24 and 25 are tested:

M6Pn-α(1,2)-Man-Ethyl-NH₂ (compo 18);

M6Pn-α(1,2)-Man-EthylSq (compo 19);

M6Pn-EthylSq (described below);

M6C-α(1,2)-Man-Ethyl-NH₂ (compo 24);

M6C-Ethyl-NH₂ (described in table 1);

M6C-α(1,2)-Man-EthylSq (compo 25);

M6C-EthylSq (described below).

Experimental Protocol

The LNCaP human prostate cancer cells are incubated with solutions ofthe analogs as described above having concentrations ranging from 10⁻³ Mto 10⁻⁷ M.

The cell survival is measured after four days of incubation by means ofan MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)test. This compound is reduced by a mitochondrial enzyme and will forman orangy-yellow colored compound which is soluble in an aqueous medium.The measurement of the optical densities thus indicates the amount oflive cells. The cell growth results were determined with respect to acontrol treated only with the carrier (solution in which the analogs arediluted).

The results obtained are represented in FIG. 1.

Conclusion:

None of the compounds tested shows any significant cytotoxicity on LNCaPhuman cells even at very high concentrations (up to 10⁻³ M).

Thus, the M6P analogs tested do not show any intrinsic toxicity forcells expressing CI-M6PR, such as LNCaP cells.

3) Functionalization of Nanoparticles for Single-Photon PhotodynamicTherapy

A compound of interest {circle around (Y)}, namely mesoporous silicananoparticles incorporating a photosensitizer of neutral porphyrin type,is then surface-functionalized with:

the phosphonate disaccharide analog M6Pn-α(1,2)-Man-EthylSq (19) and thecarboxylate disaccharide analog M6C-α(1,2)-Man-EthylSq (25);

the phosphonate monosaccharide analog M6Pn-EthylSq and the carboxylatemonosaccharide analog M6C-EthylSq.

The structure of the neutral porphyrin used for the photodynamic therapyis represented below:

Preparation of the Silica Nanoparticles Incorporating the Porphyrin

The porphyrin must bear a trialkoxysilane group which will enable it tobe incorporated into the siliceous network during the sol-gel processdescribed below. For this, the porphyrin is reacted with3-mercaptopropyltrimethoxysilane in methanol at ambient temperature for12 h.

CTAB (cetyltrimethylammonium bromide) is dissolved in a 0.2 M sodiumhydroxide solution. Micelles will thus form in the solution. Thesilylated porphyrin and then TEOS (tetraethyl orthosilicate) are added.The reaction medium is quickly diluted with a large volume of water inorder to lower the pH to 8-8.5 and to initiate condensation. After 6min, the reaction medium is neutralized with a 0.2 M HCl solution. Thenanoparticles are obtained after steps of washing with ammonium nitratein order to remove the CTAB contained in the pores.

During the synthesis of the nanoparticles, the reaction times and alsothe pH must be meticulously controlled in order to control the size ofthe nanoparticles formed. The nanoparticles thus obtained have ahydrodynamic diameter of approximately 200 nm and contain 11 μg ofporphyrin per gram of nanoparticle.

Functionalization of the Nanoparticle Surface

These nanoparticles are then surface-functionalized with the carboxylatemonosaccharide analog M6CEthylSq and the phosphonate monosaccharideanalog M6PnEthylSq and the carboxylate disaccharide analogM6C-α(1,2)-Man-EthylSq (25) and the phosphonate disaccharide analogM6Pn-α(1,2)-Man-EthylSq (19).

The functionalization is carried out in two steps; the first stepconsists in introducing amine functions onto the surface of thenanoparticles which, in the second step, will replace the ethoxy grouppresent on the squarate of the arm of the analog. The nanoparticles arereacted with aminopropyltriethoxysilane (APTS) in a water/ethanolmixture (2:1). The pH of the reaction medium is adjusted to 6 by addinga 0.2 M HCl solution and then stirred at ambient temperature for 20 h.The nanoparticles are obtained after several steps of washing withethanol, followed by centrifugations.

For the coupling of the carboxylate analogs (M6CEthylSq andM6C-α(1,2)-Man-EthylSq (25)), the analog was dissolved in anethanol/water mixture (1:1) in the presence of the nanoparticles andthen the suspension was heated at 50° C. for 12 h.

It should be noted that triethylamine can be added in order to maintainthe pH at 8-9 and to promote coupling of the analogs to thenanoparticles.

After steps of washing with water and with ethanol, the nanoparticlesfunctionalized with the carboxylate analogs are obtained.

The coupling (grafting) of the carboxylate disaccharide analogM6C-α(1,2)-Man-EthylSq (25) with the silica nanoparticles incorporatingporphyrin (represented by a star) is represented below:

A conjugate of formula (III) is thus obtained.

Since the squarate group can be observed by UV, the amount ofmonosaccharides and disaccharides was assayed by UV/visiblespectroscopy.

Table 2 below represents the amount of porphyrin incorporated (in μg/g)and the amount of monosaccharide or disaccharide (in μmol/g) graftedonto the nanoparticles.

The abbreviation “MSN” represents the mesoporous silica nanoparticlesalone, without grafting with the monosaccharide or the disaccharide.

The M6CEthylSq, M6C-α(1,2)-Man-EthylSq (25) and M6Pn-α(1,2)-Man-EthylSq(19) analogs grafted with the mesoporous silica nanoparticles arerepresented by MSN-M6C-EthylSq; MSN-M6C-α(1,2)-Man-EthylSq andMSN-M6Pn-α(1,2)-Man-EthylSq. These compounds are conjugatescorresponding to the general formula (III).

TABLE 2 Amount of Amount of porphyrin mono- or disaccharideNanoparticles (μmol/g) (μmol/g) MSN 11 μmol/g — MSN-M6C-EthylSq 11μmol/g 351 μmol/g MSN-M6C-α(1,2)-Man-EthylSq 11 μmol/g 329 μmol/gMSN-M6Pn-α(1,2)-Man-EthylSq. 11 μmol/g 167 μmol/g

In Vitro Evaluation in Photodynamic Therapy

The MSN-M6C-EthylSq and MSN-M6C-α(1,2)-Man-EthylSq carboxylateconjugates of formula (III) were used in in vitro PDT experiments onLNCaP prostate cancer cells which overexpress CI-M6PR.

The LNCaP cells were incubated with 80 μg·mol⁻¹ of nanoparticles alone(MSN) or with 80 μg·mol⁻¹ of nanoparticles functionalized with the mono-or disaccharide (i.e. with 80 μg·mol⁻¹ of MSN-M6C-EthylSq andMSN-M6C-α(1,2)-Man-EthylSq conjugates) for 3, 6, 9 or 18 h, thenirradiated for 20 min with a laser at 650 nm (3 mW, 11.25 J·cm⁻²).

The MTS cell survival test was carried out 48 h after the irradiation.

After incubation for three hours, a very slight increase in thephototoxic effect of the nanoparticles functionalized with the mono- ordisaccharides is observed.

When the incubation time is increased to 6 h, it is noted that thefunctionalization with a carboxylate disaccharide brings a strongimprovement in the phototoxic effect, with 73% cell death compared with35% for the carboxylate monosaccharide. This is linked to theimprovement in the recognition of CI-M6PRs resulting in a fasterinternalization of the nanoparticles (functionalized with adisaccharide) into the cells.

These results demonstrate the advantage of the functionalization of thenanoparticles with disaccharide analogs, making it possible both toreduce the incubation time and to considerably increase the phototoxiceffect of the nano-tools.

If the incubation time is increased to 9 h, the cell death obtained forthe cells treated with the nanoparticles functionalized with thedisaccharide reaches 81% compared with 55% for the nanoparticlesfunctionalized with the monosaccharide.

In the case of an incubation for 18 h, an improvement in the phototoxiceffect is observed for the nanoparticles functionalized with themonosaccharide carboxylate analogs compared with the effect of thesesame nanoparticles after an incubation of 9 h.

On the other hand, the effect of the disaccharide nanoparticles is notincreased at 18 h since this effect is already at its maximum after 9 hof incubation.

The phototoxic effects of the nanoparticles alone (MSN) and thenanoparticles functionalized with the carboxylate monosaccharide(MSN-M6C-EthylSq) and carboxylate disaccharide(MSN-M6C-α(1,2)-Man-EthylSq) at the various incubation times arereproduced in FIG. 2.

Conclusion

The functionalization of nanoparticles with dimannosides that areanalogs of M6P thus makes it possible to very significantly increase thephototoxic efficacy compared with the nanoparticles grafted with themonosaccharide analogs, while at the same time reducing the incubationtime. This increase in efficacy associated with the affinity for CI-M6PRcan be determined for the diagnostic or therapeutic application of thecompounds of interest {circle around (Y)} (nanoparticles, glycoproteins,small molecules, etc.), the metabolic clearance of which in human beingscan be rapid.

EXAMPLE 3

Synthesis of Compounds of Formula (I)

Preparation of Disaccharides 26, 27 and 28 Corresponding to the Formula(I) in which X Represents the Phosphonate Group

The process for preparing disaccharides 26, 27 and 28 corresponding tothe formula (I) in which X represents the phosphonate group isillustrated below:

The beginning of the synthesis of the phosphonate disaccharide 28 offormula (I) is identical to that of the phosphonate disaccharide 19,namely the phosphonate 15 is obtained from the alcohol 12 in the sameway as described above.

In order to synthesize the phosphonate disaccharide 28 of formula (I),the aldehyde 13 is reacted with tetraethyl methylenediphosphonate,deprotonated beforehand with sodium hydride, so as to form the compound14 with a yield of 75% over two steps. During a catalytic hydrogenationstep, in the presence of palladium-on-carbon and for which the hydrogenis generated in situ by gradual addition of triethylsilane, the doublebond and also the three benzyls are reduced and the trimethylsilylgroups are hydrolyzed. The compound 15, is thus obtained, withoutpurification, with a quantitative yield. The bromine atom is thenreplaced with N-hydroxyphthalimide so as to form the intermediate 26with a yield of 68%. The alcohol functions are then protected withtrimethylsilyl chloride so as to give the compound 27 with a yield of88%. The intermediate 27 thus obtained, without purification, is thenreacted with trimethylsilyl chloride and sodium iodide so as to form thebis(trimethylsilyl) phosphonate by a Rabinowitz type reaction. Thisintermediate is then converted into phosphonic acid through the actionof hydrazine monohydrate in methanol which will also displace thetrimethylsilyl groups present on the secondary alcohols of the sugar andreact with the hydroxyphthalimide in order to unmask the oxyaminefunction. The compound 28 deprotected on the phosphonate part, and onthe alcohols of the two sugars, and bearing in the anomeric position theoxyamine function, required for the coupling with the enzyme, is thusobtained in a single step, with a yield of 64% over two steps.

Conditions and reagents: (i) Dess-Martin periodinane, DCM, AT, 4 h (ii)tetraethyl methylenediphosphonate, NaH, THF, AT, 45 min; (iii) Pd/C,Et₃SiH, MeOH; (iv) N-hydroxyphthalimide, NaH, DMF, 65° C., 20 h; (v)TMSCl, NEt₃, DCM, DMF, AT, 24 h; (vi)-a TMSCl, NaI, Et₃N, ACN, 35° C., 4h; (vi)-b N₂H₄.H₂O, MeOH, AT, overnight.

Experimental Section

Preparation of 2-N-oxyphthalimidoethyl6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside26

177 mg (2.2 eq, 5.3 mmol) of NaH are suspended in 80 ml of DMF. 783 mg(2 eq, 4.8 mmol) of N-hydroxyphthalimide are added (red solution). Afterstirring for one hour at 65° C. (dark red solution), 1.4 g (1 eq, 2.4mmol) of the compound 15 dissolved in 20 ml of DMF are added. After 20 hat 65° C., the solution is cooled and the DMF is evaporated off underreduced pressure. The crude product obtained is purified on a silica gelcolumn with an EtOAc/MeOH gradient (95:5→80:20) so as to give thecompound 26 with a yield of 68% (1.08, 1.62 mmol).C₂₇H₄₀NO₁₆P  Chemical formula 26:

Molar mass: 665.58 g·mol⁻¹

R_(f): 0.6 [EtOAc/MeOH (70:30)]

Preparation of 2-N-oxyphthalimidoethyl2,3,4-tri-O-trimethylsilyl-6-deoxy-6-diethoxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-trimethylsilyl-α-D-mannopyranoside27

1.05 g (1 eq, 1.6 mmol) of the compound 26 are dissolved in 10 ml offreshly distilled DCM and 6.6 ml (47.3 mmol, 30 eq) of triethylamine.The starting product is not completely soluble. 2.4 ml (18.9 mmol, 12eq) of trimethylsilyl chloride are added dropwise. The solution isstirred at ambient temperature for 25 h and then evaporated. During thereaction, the solution becomes brown. The brown residue is dissolved incyclohexane and then filtered through celite. The filtrate isconcentrated so as to give the compound 27 with a yield of 88% (1.52 g;1.38 mmol) which is used directly for the next step.C₄₅H₈₈NO₁₆Si₆  Chemical formula 27:

Molar mass: 1098.67 g·mol⁻¹

R_(f): 0.57 [DCM/Et₂O (90:10)]

Preparation of aminooxyethyl6-deoxy-6-dihydroxyphosphinylmethylene-α-D-mannopyranosyl-(1→2)-D-mannopyranoside28

Et₃N (7.2 eq, 0.34 ml, 2.4 mmol), TMSCl (7 eq, 0.296 ml, 2.34 mmol) andNaI (7 eq, 350 mg, 2.34 mmol) are added, under nitrogen atmosphere, to asolution of compound 27 (1 eq, 367 mg, 0.33 mmol) in 11 ml of anhydrousCH₃CN. After 4 h at ambient temperature, the stirring is stopped. Thesupernatant is drawn off with a tube and the residual salt is rinsedwith anhydrous CH₃CN (2×3 ml) and then the solvent is drawn off with atube. The solvent is evaporated off and the residue obtained is taken upin 7.5 ml of anhydrous methanol containing hydrazine (12 eq, 4.01 mmol,0.194 ml). The reaction is left to stir overnight. A white precipitateis formed. The residue obtained after evaporation is purified bychromatography on a silica gel column (eluent: 5:4:1isopropanol/NH₄OH/H₂O) so as to give the compound 28 with a yield of 64%(103 mg, 0.22 mmol).C₁₅H₃₀NO₁₄P  Chemical formula 28:

Exact weight: 479.37 g·mol⁻¹

R_(f): 0.33 [isopropanol/NH₄OH/H₂O (5:4:1)]

MS, ESI⁺ m/z: 480.3 [M+H]⁺

EXAMPLE 4

Study of the Disaccharide Analog 28 of the Invention, Corresponding tothe General Formula (I)

1) Functionalization of a Lysosomal Enzyme, Acid Alpha-Glucosidase(GAA), with the Disaccharide Analog (28)

A compound of interest {circle around (Y)}, namely a lysosomal enzyme,acid alpha-glucosidase (GAA), which can be used for the treatment ofPomp disease or glycogenosis type II⁷⁻⁸, is functionalized on theoligosaccharide chains with the phosphonate disaccharide analogM6Pn-α(1,2)-Man-Ethyloxyamino (28). Pomp disease is representative ofthe majority of the 53 different lysosomal diseases for which theefficacy of treatment by enzyme replacement therapy depends onCI-M6PR⁷⁻⁹. Specifically, this receptor plays a key role in cellinternalization of intravenously injected recombinant enzymes and in theintracellular routing thereof to the lysosomes.

Experimental Protocol:

Recombinant human GAA (rhGAA) is produced in the system of CHOeukaryotic cells capable of producing M6P at the end of the glycosylatedchains and is purified from the culture medium. The molecular weight ofthe enzyme is approximately 110 000 Da and it is recognized by aspecific anti-human GAA antibody (anti-LYAG, Genetex). The recombinanthuman GAA can also be produced in the baculovirus/Sf9 insect cell systemwhich does not produce M6P residues and which produces mannosylatedoligosaccharide chains.

The rhGAA is then functionalized on the oligosaccharide chains with thephosphonate disaccharide analogs M6Pn-α(1,2)-Man-Ethyloxyamino (28). Thefunctionalization is carried out in two steps: the first step consistsin generating aldehyde functions by oxidation of the mannoses which, inthe second step, will react with the oxyamine functions present on theethyloxyamino arm of the analog (28). The enzymes are oxidized by 1 mMNaIO₄ for 30 min at 4° C. and then reacted with the disaccharide analogs(100 equivalents for 1 enzyme) for 2 h at 25° C. The functionalizedenzymes are obtained after dialysis in order to remove the unreactedanalogs.

The coupling (grafting) of the phosphonate disaccharide analogM6Pn-α(1,2)-Man-Ethyloxyamino (28) with the enzymes is representedbelow:

The oxidation of the sialic acid is carried out using sodium periodate.

The oxidation of the mannose is carried out using sodium periodate.

2) Affinity for CI-M6PR of the rhGAA Functionalized withM6Pn-α(1,2)-Man-Ethyloxyamino (28)

The conjugate of formula (III), namely the rhGAA functionalized with thephosphonate disaccharide analog M6Pn-α(1,2)-Man-Ethyloxyamino (28), isdenoted hereinafter rhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino (28).

The conjugate of formula (III) is evaluated for its affinity withCI-M6PR. The binding affinity of rhGAA and of therhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino conjugate (28) was evaluated bymeans of a competitive binding technique with CI-M6PR described inexample 2 and compared with the affinity of M6P.

The data indicate a strong affinity forrhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino (28) (conjugate of formula (III))corresponding to a 50% inhibitory concentration (IC₅₀) of 0.55 10⁻⁷ M,while the affinity of the rhGAA was 3×10⁻⁷ M, i.e. 5.5 times lower. Bycomparison, the affinity of M6P is 3×10⁻⁵ M in these same experiments.This result indicates the efficient coupling of several dimannosideanalogs of M6P on the glycosylated chains of the enzyme.

3) Internalization of the rhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino Conjugate(28) (Conjugate of Formula (III)) in the Myoblasts of Patients Sufferingfrom the Adult Form of Pomp Disease (FIG. 3)

Experimental Protocol:

The myoblast-type cells originating from an adult patient suffering fromPomp disease are maintained in primary culture.

The catalytic activity of GAA is measured in the cell extracts using thesynthetic substrate 4-methylumbelliferyl-α-D-glucopyranoside (4-MUG).This substrate and the molecular weight standards are obtained fromSigma. The GAA activity in the cell extracts (20 μl) is measured in areaction volume of 75 μl containing 50 mM citric acid, 115 mM K₂HPO₄,110 mM KCl, 10 mM NaCl, pH 5.0, with 6 mM 4-MUG, for 10 min at ambienttemperature. The reaction is stopped with 75 μl of 0.1 M Tris HCl, pH 8.The fluorescence is read with an excitation filter at 355 nm and anemission filter at 460 nm in 96-well plates, and compared to a standardcurve obtained with 4-methylumbelliferone. The catalytic activity isexpressed per mg of protein. Mean±standard deviation.

It emerges from FIG. 3 that the grafting of the disaccharidessignificantly increases the cell penetration of the GAA, theconcentration of which is measured through its catalytic activity on the4-MUG substrate.

Conclusion:

The product of interest, rhGAA functionalized by the addition ofdisaccharides (28), is more effective in the treatment of an enzymaticdeficiency associated with Pomp disease.

4) Study of the Process of Cellular Maturation in Myoblasts from anAdult Patient Suffering from Pomp Disease of therhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino Conjugate (28) (Conjugate of Formula(III)) (FIG. 4)

The cellular maturation of GAA¹⁰ is carried out in the endosomes andlysosomes by several specific enzymatic cleavages which successivelyconvert the inactive precursor of 110 kDa into intermediate forms of 95kDa and 76 kDa, then into a mature and active form of 60-70 kDa in thelysosomes¹⁰.

The myoblasts are incubated for 48 h in the presence of 50 nM rhGAA orof the rhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino conjugate (28) (conjugate offormula (III)), then lysed and the proteins are extracted. The cellularmaturation of the internalized GAA enzyme in the myoblasts of patientssuffering from Pomp disease is studied by the SDS polyacrylamide gelseparation technique, followed by immunodetection of the GAA by Westernblot with an anti-human GAA antibody (anti-LYAG, Genetex) or ananti-human actin antibody (Invitrogen). The actin is revealed as acontrol protein indicating that equivalent amounts of proteins areloaded onto the gel. This experiment is representative among twoexperiments. The thick arrow in FIG. 4 indicates the intermediate formof GAA. The black arrow indicates the mature form of GAA (60-70 kDa).

The mature form of 60 kDa is quantified by considering the band presentin the myoblasts originating from a healthy subject to be 100%. Theexpression of GAA 60 kDa in the patient myoblasts corresponds to 1% ofthe GAA of a healthy subject and is not increased in the myoblaststreated with rhGAA. Conversely, the treatment with therhGAA-M6Pn-α(1,2)-Man-Ethyloxyamino conjugate (28) increases the 60-70kDa band to 47% of the GAA of a healthy subject. These results indicatethat the grafting of the disaccharide analogs very strongly promotes theprocess of intracellular maturation of the rhGAA, resulting in itsactive form of 60-70 kDa. This activity is important for an applicationin the treatment of Pomp disease by enzyme therapy, but can also beapplicable to all lysosomal diseases.

Conclusion

The functionalization of a lysosomal enzyme with modified dimannosidesthus makes it possible to very significantly increase the cellular entryof this enzyme compared with the non-grafted enzyme in cells from apatient suffering from Pomp disease. The functionalization with thedimannoside analogs of M6P makes it possible to facilitate thematuration of the enzyme inside the endosomes and lysosomes comparedwith the non-functionalized enzyme. This increase in effectivenessassociated with the affinity for CI-M6PR can be determined for thediagnostic or therapeutic application of glycoproteins, in particularfor enzyme replacement therapy used for lysosomal overload diseases⁷⁻⁹.

LITERATURE REFERENCES

-   1. FR patent application No. 14 50588 filed on Jan. 23, 2014;-   2. Vidal S et al., Bioorg Med Chem. 10, 4051, (2002);-   3. Jeanjean A et al., Bioorg Med Chem. 14, 2575, (2006);-   4. Jeanjean A et al., Bioorg Med Chem Lett. 18, 6240, (2008);-   5. International application PCT/EP2010/059507;-   6. Distler J J et al., J. Biol Chem, 32, 21687, (1991);-   7. Desnick, R J, Schuchman E H, Nat Rev Genet 3, 954, (2002);-   8. Van der Ploeg A T, Reuser A J, Lancet, 372, 1342, (2008);-   9. Hollak, C E, Wijburg F A, J Inherit Metab Dis, 37, 587, (2014);-   10. Moreland R J et al., Gene, 491, 25, (2012).

The invention claimed is:
 1. A compound having the general formula (I):

in which: n is an integer ranging from 1 to 3,

represents a single bond or a double bond, each of the P¹ represents,independently of one another, H, or an acid-labile, base-labile,hydrogen-labile, photo-labile or halogen-labile protecting group, inparticular chosen from (CH₃)₃Si—; tBuMe₂Si—; C₆H₅CH₂—; 4-CH₃OC₆H₅CH₂—;o-NO₂C₆H₅CH₂—; CH₃CO—; C₆H₅CO— or CF₃CO—; X represents: when

is a single bond: the phosphonate group:

the fluorophosphonate group:

the difluorophosphonate group:

the carboxylate group:

the malonate group:

when

is a double bond: the phosphonate group:

the carboxylate group:

with Z representing, independently of one another, H; a C₁₋₅ alkyl;CF₃CH₂—; C₆H₅CH₂—; C₆H₅—; (CH₃)₃Si—; an alkali metal chosen from Na, Lior K; an ammonium NH₄; A represents a divalent radical chosen from —O—,—S—, —NH—, —CH₂—; L represents: —H; —NH₂; —(CH₂)_(n1)—CH═CH₂ or—(CH₂)_(n1)—C≡CH with n₁ representing an integer ranging from 0 to 4,then in each of these cases, L₁ is absent, a substituted orunsubstituted, linear or branched, saturated divalent hydrocarbon-basedradical having from 1 to 30 carbon atoms, a substituted orunsubstituted, linear or branched, unsaturated divalenthydrocarbon-based radical having from 2 to 30 carbon atoms, a saturatedor unsaturated divalent hydrocarbon-based radical as defined above, inwhich one or more —CH₂—, —CH═CH— and/or —C≡C— groups of the saturated orunsaturated hydrocarbon-based radical is (are) replaced, independentlyof one another, with an —O— group; —NH— group; —NR₁— group with R₁representing a C₁-C₅ alkyl; —S— group; —CO—NH— group; —NH—CO—O— group;—O—N═CH— group; —CO—NH—N═CH— group; a substituted or unsubstituted,saturated or unsaturated, cyclic or heterocyclic system; L₁ represents:—(CH₂)_(n1)—CH═CH₂; —(CH₂)_(n1)—C≡CH; —(CH₂)_(n1)—N₃; —(CH₂)_(n1)—SH;—(CH₂)_(n1)—NH₂; —(CH₂)_(n1)—N═C═O; —(CH₂)_(n1)—N═C═S; —(CH₂)_(n1)—NHR₁;—(CH₂)_(n1)—NR₁R₂; —(CH₂)_(n1)-A₁-NH₂; —(CH₂)_(n1)-A₁-NHR₁;—(CH₂)_(n1)-A₁-NR₁R₂; —(CH₂)_(n1)—NHCO—CH₂Hal; —(CH₂)_(n1)—COZ₁;—(CH₂)_(n1)-A₁COZ₁; —(CH₂)_(n1)—O—N═CH₂; —(CH₂)_(n1)—CO—NH—N═CH₂;—(CH₂)_(n1)—H; a substituted or unsubstituted, saturated or unsaturated,cyclic or heterocyclic system; a halogen chosen from F, Cl, Br or I;with n₁ as defined above, R₁ and R₂ representing, independently of oneanother, a C₁-C₅ alkyl, A₁ representing —O—, —NH—, Hal representing Cl,Br or I; Z₁ representing —OH, —OR₁, —NHR₁, —NH—NH₂, —NH—NHR₁, —NH—NR₁R₂,with R₁ and R₂ as defined above, a halogen chosen from F, Cl, Br or I.2. The compound as claimed in claim 1, wherein: L represents: —NH₂,—(CH₂)_(n1)—CH═CH₂ or —(CH₂)_(n1)—C≡CH, with n₁ as defined in claim 1,then in each of these cases, L₁ is absent, a substituted orunsubstituted, linear or branched, saturated divalent hydrocarbon-basedradical having from 1 to 10 carbon atoms, a substituted orunsubstituted, linear or branched, unsaturated divalenthydrocarbon-based radical having from 2 to 10 carbon atoms, a saturatedor unsaturated, divalent hydrocarbon-based radical as defined above, inwhich one or more —CH₂—, —CH═CH— and/or —C≡C— groups of the saturated orunsaturated hydrocarbon-based radical is (are) replaced, independentlyof one another, with: an —O— group; —NH— group; —S— group; —CO—NH—group; —NH—CO—O— group; and/or a cyclic or heterocyclic system chosenfrom:

L₁ represents: —(CH₂)_(n1)—CH═CH₂; —(CH₂)_(n1)—C≡CH; —(CH₂)_(n1)—N₃;—(CH₂)_(n1)—SH; —(CH₂)_(n1)—NH₂; —(CH₂)_(n1)—N═C═O; —(CH₂)_(n1)—N═C═S;—(CH₂)_(n1)—O—NH₂; —(CH₂)_(n1)—NHCO—CH₂Hal; —(CH₂)_(n1)—COOH;—(CH₂)_(n1)—COOR₁; —(CH₂)_(n1)—CO—NH—NH₂; —(CH₂)_(n1)—H; a halogenchosen from F, Cl, Br or I; a cyclic or heterocyclic system chosen from:

with n₁, R₁ and Hal as defined in claim
 1. 3. The compound as claimed inclaim 1, wherein: the substituent L is chosen from: —CH═CH₂, —C≡CH or—NH₂, then in each of these cases, L₁ is absent; —CH₂—; —CH₂—CH₂—;—(CH₂)₃—; —(CH₂)₄—; —(CH₂)₅—; —CH₂—CH₂—O—CH₂—CH₂—;—(CH₂—CH₂—O)₂—CH₂—CH₂—; —CH₂—CH₂—S—CH₂—CH₂—;—CH₂—CH₂—S—CH₂—CH₂—O—CH₂—CH₂—;

and the substituent L₁ is chosen from: —CH═CH₂; —C≡CH; —N₃; —SH; —NH₂;—N═C═O; —N═C═S; —O—NH₂; —NHCO—CH₂Cl; —COOH; —COOR₁; —CO—NH—NH₂, ahalogen chosen from F, Cl, Br or I, a cyclic or heterocyclic systemchosen from:

with R₁ as defined in claim
 1. 4. A process for preparing a compound offormula (I) as claimed in claim 1, wherein the starting compound used isa compound corresponding to the formula (II):

in which P¹, A, L, L₁ and n are as defined in claim
 1. 5. A conjugatehaving the general formula (III):

in which P¹, X, n, A and L are as defined in claim 1, L′₁ represents thesubstituent L₁ as defined in claim 1 involved in a covalent bond with afunctional group borne by a compound of interest

said compound of interest

being chosen from enzymes, nanoparticles, proteins, antibodies orcytotoxic agents.
 6. The conjugate as claimed in claim 5, wherein saidconjugate has an IC₅₀ affinity for the cation-independent mannose6-phosphate receptor (CI-M6PR) of at least 10⁻⁵ M, and preferablyranging from 10⁻⁶ to 10⁻⁹ M.
 7. The conjugate as claimed in claim 5,wherein the compound of interest

is a lysosomal enzyme or a nanoparticle.
 8. A method for therapeutictreatment of a human or animal, wherein the therapeutic treatment isselected from enzyme replacement therapy, photodynamic therapy or cancertreatment comprising administering the conjugate of formula (IIIa)

to the human or animal.
 9. A method of detecting diseases or ailmentsassociated with an increase or a decrease in CI-M6PR expression in ahuman or animal comprising administering the conjugate of claim 5 to thehuman or animal and detecting binding of the conjugate to CI-M6PR.
 10. Amethod of forming at least one covalent bond with at least onefunctional group borne by a compound of interest

said compound of interest

being chosen from enzymes, nanoparticles, proteins, antibodies orcytotoxic agents comprising contacting the compound of claim 2 with anenzyme, nanoparticle, protein, antibody or cytotoxic agent.
 11. Themethod as claimed in claim 10, wherein n′ compound(s) of formula (I) arepresent for forming n′ covalent bond(s) with n′ functional group(s) ofthe compound of interest

with n′ being an integer ranging from 1 to
 1000. 12. The method claimedin claim 10, for forming the conjugate of general formula (IIIa):

in which P¹, X, n, A, L, L′₁ and

are as defined in claim 5, n′ is as defined in claim
 11. 13. A processfor preparing a conjugate of formula (III)

or a conjugate of formula (IIIa)

comprising reacting: at least one functional group borne by a compoundof interest

said compound of interest being chosen from enzymes, nanoparticles,proteins, antibodies or cytotoxic agents, with at least one compoundcorresponding to the formula (I):

in which P¹, X, A and n are as defined in claim 1, L and L₁ are asdefined in claim 2 or 3.